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Long-term results of short segmental syngeneic small intestinal transplantation: Comparison of jejunal and ileal grafts
Long-Term Results Transplantation: By Muhammad
of Short Segmental Syngeneic Small Intestinal Comparison of Jejunal and Ileal Grafts
S. Rahman,
Tomoaki
Taguchi, Makoto Nakao, Takaharu Fukuoka, Japan
l A short-segment intestinal graft is favorable to reduce the rate of rejection and the incidence of graft-versus-host disease in recipients of small intestine transplantation. To determine whether a jejunal or an ileal graft is preferable with respect to intestinal morphology and function, syngeneic two-step small intestinal transplantations were performed using male Lewis rats (RTI’). They were divided into two groups according to the small intestine donor site (group 1 received 10 cm of jejunum; group 2 received 10 cm of ileum). There was no significant difference in the survival rate or weight gain between the two groups. Nearly all the hematologic findings, serum nutritional parameters, and results of liver function tests were normal for both groups. The only difference was that the serum total bile acid level was significantly higher in group 2. Fifty weeks after transplantation, the graft mucosa showed normal architecture, with adaptive hyperplasia of villi and crypt noted through histological study. The villus height of group 1 was 595 ? 64 pm (control, 452 f 67 pm); that of group 2 was 732 2 53 pm (control, 217 ir 20 pm). The crypt depth of group 1 was 228 + 35 pm (control, 165 f 24 pm); that of group 2 was 320 2 19 urn (control, 102 f 19 pm). These compensatory changes were more pronounced in group 2. The authors conclude that, on the basis of long-term functional capacity, there was no significant difference between jejunal and ileal grafts, and that both segments were suitable for transplantation. However, the ileal graft was considered to be better with respect to morphological adaptation. Copyright o 1996 by WA Saunders Company
Yamada,
and Sachiyo
Suita
with SBS. Small intestinal segments can be obtained from living-related donors, which not only increases the number of available grafts but also the chances of compatibility.4 Recent reports have shown that there is no significant difference in the immunologic response between ileal and jejunal grafts,5,6 and have suggested that, in light of rejection and graft-versushost disease (GVHD), the small intestine donor site should be selected on the basis of its distinctive functional capacity. 5 Clinical cases of SIT, using effective immunosuppressants such as cyclosporine7-9 or FK-506,10J1 have been reported recently from several institutions. However, the long-term survival rate was not as good as for other organs. In addition, to overcome the immunologic problems, it is also important to select the most suitable part of the intestine to be used in transplantation. The purpose of the present study, in which we compared the long-term morphological and functional results achieved with short segment jejunal and ileal isografts, was to determine which graft would be preferable for transplantation. MATERIALS
AND METHODS
Animals and Groups INDEX WORDS: Small intestine, ileum, total bile acid, villus height.
transplantation,
jejunum,
T
HE IMPROVEMENT in total parenteral nutrition (TPN) and its modified versions, such as home parenteral nutrition, have significantly improved the quality of life of patients with short bowel syndrome (SBS). However, a number of serious complications (eg, catheter-related sepsis, metabolic imbalance, depressive reaction) are believed to be inevitable with long-term use of TPN.rW3 Therefore, small intestinal transplantation (SIT) is becoming more popular as the radical treatment for patients
From the Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Supported in part by a Grant-in-Aid for General Scientijic Research (C) and by a Grant for Pediatric Research (6C-02) both from the Ministry of Health and Welfare, Japan. Address reprint requests to Muhammad S. Rahman, MD, Department of Pediattic Surgery, Faculty of Medicine, Kyushu University, 3-I-1, Maidashi, Higashi-ku, Fukuoka 812-82, Japan. Copyright o 1996 by WB. Saunders Company 0022-346819613107-0009$03.00/0 908
Syngeneic SIT was performed using male Lewis (RTl’) rats (Charles River Co, Ltd, Yokohama, Japan), weighing 200 to 250 g. They were divided into two groups, according to the small intestine donor site. Group 1 (n = 8) received a lo-cm segment of proximal jejunum from the ligament of Treitz. Group 2 (n = 9) received a lo-cm segment of terminal ileum.
Transplantation Procedures The rats, both donor and recipient, were fasted overnight but allowed to drink water ad libitum. Orthotopic segmental SIT was performed in two steps, according to a modification of the methods first described by Monchick and Russell.12 Briefly, the donor, under sedation with ether, was anesthetized by intraperitoneal administration of pentobarbital sodium (40 mgikg body weight) with atropine sulfate (0.05 mgikg body weight). Chloral hydrate (160 mgikg body weight) was added by intravenous or intraperitoneal administration, as necessary. Under operative microscopy (Wild M715; Wild Leitz, Heerbrugg, Switzerland), a lo-cm segment of proximal jejunum, or distal ileum, from the donor was isolated on a pedicle of the aorta and portal vein. Immediately after graft harvesting, the intestinal lumen was irrigated with cold (4°C) lactated Ringer’s solution (LR) containing 1 mg/mL Amicamycin sulfate (Meiji Pharmaceutical, Tokyo, Japan); the aorta of the graft was flushed with cold LR containing 10 UlmL of heparin. The graft was preserved in cold LR until transplanted. The recipient was anesthetized in the same fashion as the donor, and an intravenous infusion of LR was administered, at a rate of 10 mL/h, via the cannulated tail JournalofPediatric
Surgery,
Vol31,
No 7 (July),
1996:
pp 908-911
SEGMENTAL
INTESTINAL
vein or the jugular vein. The intestinal isograft was revascularized immediately by performing end-to-side anastomosis of the donor aorta to the recipient infrarenal aorta, and of the donor portal vein to the recipient inferior vena cava, using 9-O nylon sutures. Both ends of the graft were exteriorized through the right abdominal wall, as a Thiry-Vella loop. The entire native small intestine, from the ligament of Treitz to 0.5 cm proximal to the ileo-cecal valve, was replaced with the graft 7 to 10 days after transplantation. The graft was placed in functional gastrointestinal continuity with two end-to-end intestinal anastomoses between the remaining native bowel and the isograft. Deaths owing to technical failure (approximately 10%) that occurred within 72 hours of operation were excluded from the study.
Follow- Up Studies Immediately after transplantation, the recipients were allowed free access to water and standard rat food. After inserting the graft into gastrointestinal continuity, the animals received water ad libitum. A normal laboratory diet was resumed 48 hours later. Evaluation of the general health status of the animals included examination of appearance, posture, and feeding habits. The weight of the animals was examined at l-week intervals.
Biochemical
909
TRANSPLANTATION
Studies
Blood samples (2 mL) were obtained from each animal 1,4, and 50 weeks after transplantation. These samples were taken from the tail vein. Ether was used for sedation. Complete blood count (CBC) (RBC, hemoglobin, hematocrit, WBC, platelet), serum nutritional parameters (total protein, albumin, cholesterol), and liver function test results (glutamic pyruvic transaminase [GPT], bilirubin, total bile acid) were studied.
Histological Examination Five rats from each group were killed 50 weeks after transplantation and used for histological examination. Biopsy specimens of the transplanted intestine were obtained and fixed in 10% formaldehyde. After being embedded in paraffin, circular samples were stained with hematoxylin and eosin and examined by light microscopy. Five samples (each about 1 cm long) of both jejunum and ileum were obtained at the time of donor operation and were used as controls for the histological study.
Statistical Analysis All data were expressed as mean 2 standard deviation. Statistical analyses were performed using the generalized Wilcoxon, Cox-Mantel, and Student’s f tests. Probability values of less than .05 were considered significant.
%
Group 2(n=9)
IOO-
I
BO-
g 2 602 s 40-
Group
zo0
20
0
Wilcoxon and Cox-Mantel tests (Fig l), there was no significant difference in the survival rates of the two groups.
60
There
were
80
120
week
differences
be-
100
no significant
tion period. There was no statistically significant difference in weight gain between the two groups. Biochemical Evaluations The RBC, WBC, serum total protein, and GPT did not differ significantly between the two groups. The results of the hematologic study are shown in Table 1; those of the biochemical study appear in Table 2. The results were normal except for the significantly higher (I’ < .05) serum total bile acid level in group 2 (Table 2, Fig 2). The increased level of bile acid was first noted 1 week posttransplantation, and was confirmed 4 and 50 weeks after transplantation. Histological Findings The mucosal villus height of the proximal jejunum and of the terminal ileum in the control study was 452 4 67 pm and 217 r 20 km, respectively (Table 3). The corresponding crypt depths were 165 2 24 km and 102 & 19 km. The mucosal villus height of the grafts in group 1 and group 2 was 595 +- 64 km and 732 ? 53 km, respectively. The extention ratios of graft villus height for the two groups (compared with controls) were 1.32 and 3.32, respectively. The corresponding mucosal crypt depths were 228 L 35 brn and 320 f 19 km. The extention ratios of crypt depth of the grafts in groups 1 and 2 (compared with controls) were 1.38 and 3.14, respectively. The extention rates of villus height and crypt depth were higher in group Table
1. Hematologic
Parameter
Hemoglobin level (g/dL)
Hematocrit
Platelet
Weight Gain All rats ate normally, remained healthy, and gained weight at a satisfactory rate throughout the observa-
40
Fig 1. Survival curve. tween the two groups.
RESULTS
Survival All but three of the rats that had transplantation had excellent results. According to the generalized
1 (x1=8)
count
Week
Group 1
1 4
12.1 T 1.6 14.9 2 0.7 14.8 2 1.7
50 1 4 50 1
(%)
(104/mm3)
4 50 NOTE. groups.
There
were
no
Findings
significant
39.9 -+ 5.4 46.5 + 1.6 45.4 + 3.3 104 2 10.2 91.6 zi 37.3 48.3
+ 16.8
differences
Group
2
12.2 c 2.0 14.6 t 0.5 16.1 CL 2.0 37.7 2 7.9 46.7 + 2.1 50.3 k 5.1 95.1 91.4 57.0 between
2 30.7 + 16.1 + 10.9 the
two
910
RAI-IMAN
Table
2. Nutritional
Parameter Albumin
level
Cholesterol
Total
Total
level
Abbreviation:
acid
level
Function
Parameters
Table
Group 1
Group 2
P Value
1 4
3.6 + 0.3 4.1 2 0.6
3.7 + 0.2 4.1 + 0.2
NS
50 1
4.0 ? 0.6 74.5 -t 14.4 77.8 84.4 0.56
-t 6.6 + 21.1 zk 0.22
NS NS NS
(mg/dL)
4 50 1
4.5 2 0.3 60.2 2 19.2 73.9 2 7.1 87.3 0.53
0.69 -t 0.37 0.54 i 0.42 14.2 c 6.8
0.51 0.55 90.7
NS NS NS
(pmol/L)
4 50 1
+ 4.6 e 0.31 r 0.13
4 50
17.9 IL 8.5 17.8 ” 13.3
101.2 73.8
zz 0.44 ‘- 63.2 A 21.8
NS c.05 <.05
25 48.7
< .05
(mg/dL)
level
Liver
Week
(g/dL)
bilirubin
bile
and
NS
Analysis
of Intestinal
Mucosa
Group 1 Villus height Control Graft (50~wk) Extension rate Crypt depth Control Graft (50-wk) Extension rate VillusCrypt ratio Control Graft
(50~wk)
Group
2
452
f 67 Pm
217
t 20 pm
595
2 64 pm 1.32
732
+ 53 pm 3.32
165 t 24 pm 228 +- 35 pm 1.38
102 * 19 pm 320 f 19 pm 3.14
2.79 2.61
2.13 2.29
NS, not significant.
2. The ratios of villus height to crypt depth of jejunum in the control group and group 1 were 2.79 and 2.61, respectively. These ratios for the control group and group 2 were 2.13 and 2.29, respectively. DISCUSSION
With respect to clinical SIT, several factors should be considered when selecting the segment and the length of donor small intestine. These are availability of the segmental graft from living-related donors (which increases the chance of compatibility), the smaller quantity of lymphoid tissue with the segmental graft (which may induce a lesser immunologic reaction), limited space in the abdominal cavity, and the capability of the segmental graft to provide the recipient’s nutritional needs. These are the principal advantages of the segmental intestinal graft over the whoIe intestinal graft. Segmental human SIT has been used in clinical practice. The jejunum, which has less lymphoid tissue and higher enzyme concentrations, has been considered the segment of choice.13 However, Revillon et al7 used a jejuno-ileal segment, and Todo et ali0 used the
~mol/L 150
3. Morphological
ET AL
0.
l$
0
:
Fig 2. Serum total bile acid level. Compared with group had a significant increase in the serum total bile acid level. level was maintained throughout the period of observation.
1, group 2 This high
entire small intestine (with or without the colon) as intestinal allografts in recent clinical transplantations. Kimura et all4 confirmed that the frequency and severity of GVHD correlated with the length of the transplanted intestine. Another advantage of segmental SIT is that lower doses of immunosuppressants are required for good graft tolerance.15 Thus, segmental SIT had many advantages and no major disadvantages. Lossing et all6 showed that GVHD and signs of rejection occurred with the same frequency in both types of intestinal segments. It also has been reported that the time of onset of rejection and GVHD was not related to the segment of transplanted small intestine.‘” Other investigators have shown no significant difference in posttransplant immunologic responses between jejunal and ileal grafts in the rat mode1.5J3 Stangel et al5 suggested that, in light of both GVHD and rejection, the selection of the portion of small intestine to be used for transplantation should be made on the basis of distinctive absorptive capacity rather than of immunologic difference. In another study, Stangel et ali7 reported that equal doses of cyclosporine did not prolong survival, nor alter the course of rejection of jejunal or ileal grafts. In a dog model, Benchimol et al6 noted no major difference between jejunal and ileal allografts with respect to GVHD or rejection. Considering all these studies, it appears that there was no significant difference in the immunologic response between jejunal and ileal grafts. Therefore, measurement of the functional and morphological capacities after transplantation are of the utmost importance in selecting the portion of intestine to be transplanted. In our study, both intestinal segments exhibited satisfactory functional performance. The eating habits of the rats were normal. Two weeks after transplantation, the bulk and consistency of their stool output were the same as those of normal rats. Regardless of the type of graft, the necessary functions of digestion and absorption were maintained. Both groups had a good survival rate and weight gain. Our results
SEGMENTAL
INTESTINAL
911
TRANSPLANTATION
showed that a transplanted segment of as short as 10 cm permitted growth and long-term survival on a normal diet. The CBC results and serum nutritional parameters were normal. There were no significant differences between the two groups. The only difference with respect to liver function was that rats with ileal grafts had a significantly higher level of serum total bile acid. This may be explained by the presence of an active transport system for bile acid in the ileum and the systemic venous drainage of the graft (partial portosystemic shunt). The diagnosis of rejection in SIT is questionable. Except for histological examination, there are few practical and effective methods for the early diagnosis of rejection. Moreover, biopsy specimens must be obtained endoscopically, and such specimens may be inadequate for diagnosis. 18,19In addition, histological changes may be best seen in submucosa and muscle tissue. However, obtaining biopsy specimens from the intestinal wall, including deeper layers, would significantly increase the risk of perforation.18 Therefore, to gain diagnostic certainty in the monitoring of allograft rejection, additional markers must be introduced along with the histological examination. The high level of serum bile acid detected in transplanted ileal grafts could act as a possible serum marker of rejection, but further experimentation is necessary.
Histologically, the grafted intestine in the present study had a normal architecture. Villus height and crypt depth were higher in the jejunum than in the ileum. However, 50 weeks after transplantation, villus height and crypt depth increased, and were higher in the ileal grafts. This indicates that the ileal mucosa has a higher capability of morphological adaptation. Our finding that the ileum possess the higher morphological adaptability is identical to that of Hanson et a1,20who studied a short bowel model in the rat. They also showed that bile, pancreatic juice, secretin, cholecystokinin, somatostatin, and gastrin were among the potential mediators of intestinal adaptive changes. However, they did not show any difference in their production between the jejunal and ileal groups. In conclusion, the similar functional performance of ileal and jejunal segments make both suitable for grafting in SIT. Morphologically, the ileal segment may be better than the jejunal segment. Further investigations are needed to determine whether the increased level of bile acid in the ileal graft can act as a serum marker of rejection when the ileum is transplanted as an allograft. ACKNOWLEDGMENT The authors are grateful remain anonymous.
to their
proofreader,
who would
like to
REFERENCES 1. Scribner BH, James JC, Christopher TG, et al: Long term total parenteral nutrition; The concept of an artificial gut. JAMA 212:457-463,197O 2. Wolfe BM, William HB, Hayashi JT, et al: Experience with home parenteral nutrition. Am J Surg 146:7-14, 1983 3. Kurkchubache AG, Smith SD, Rowe MI, et al: Catheter sepsis in short-bowel syndrome. Arch Surg 127:21-25,199Z 4. Fortner JG, Sichuk G, Litwin SD, et al: Immunological responses to an intestinal allograft with HL-A-identical donorrecipient. Transplantation 14:531-535,1972 5. Stangel MJ, Schraut WH, Moynihan HL, et al: Rejection of ileal versus jejunal allografts. Transplantation 47:424-427,1989 6. Benchimol D, Alain P, Bayer PD, et al: Jejunal versus ileal segmental allografts in the dog; Comparison of immunologic and functional results. Surgery 112:918-927,1992 7. Revillon Y, Jan D, Goulet 0, et al: Small bowel transplantation in seven children; Preservation technique. Transplantation Proc 23:2350-2351, 1991 8. Goulet 0, Revillon Y, Canioni D, et al: Two and one-halfyear follow-up after isolated cadaveric small bowel transplantation in an infant. Transplant Proc 24:1224-1225,1992 9. McAlister V, Wall W, Ghent C, et al: Successful small intestinal transplantation. Transplant Proc 24:1236-1237, 1992 10. Todo S, Tzakis A, Reyes J, et al: Small intestinal transplantation in human with or with out the colon. Transplantation 57:840848,1994 11. Hashimoto T, Zhong RZ, Bertha M, et al: Treatment with FK506 prevents rejection of rat colon allografts. Transplantation 57:1548-1554,1994
12. Monchick GJ, Russell PS: Transplantation of small bowel in the rat; Technical and immunological considerations. Surgery 70:693-701,197l 13. Pirenne J, Disilva M, Hamoir E, et al: Influence of the length of the small bowel graft on the severity of graft versus host disease. Microsurgery 11:303-308,199O 14. Kimura K, Money SR, Jaffe BM, et al: The effect of size and site of origin of intestinal grafts on rat small bowel transplantation. Surgery 101:618-622,1987 15. Kimura K, Money SR, Jaffe BM, et al: The effect of cyclosporine on varying segments of small-bowel grafts in the rat. Surgery 4:64-69,198s 16. l&sing A, Nordgren S, Cohen Z, et al: Histologic monitoring of rejection in small intestinal transplantation. Transplantation Proc 14:643-645, 1982 17. Stangel MJ, Schraut WH, Moynihan HL, et al: Effect of cyclosporine therapy in controlling the rejection of ileal versus jejunal allografts. Transplant Int 3:149-155, 1990 18. Schmid T, Oberhuber G, Korozsi G, et al: Histologic pattern of small bowel allograft rejection in the rat; Mucosal biopsies do not provide sufficient information. Gastroenterology 96:1529-1532, 1989 19. Millard PR, Dennison A, Hughes DA, et al: Morphology of intestinal allograft rejection and the inadequacy of mucosal biopsy in its recognition. Br J Exp Path01 67:687-698, 1986 20. Hanson WR, Osborne JW: Compensation by the residual intestine after intestinal resection in the rat. Gastroenterology 72:701-705,1977
of Short Segmental Syngeneic Small Intestinal Comparison of Jejunal and Ileal Grafts
S. Rahman,
Tomoaki
Taguchi, Makoto Nakao, Takaharu Fukuoka, Japan
l A short-segment intestinal graft is favorable to reduce the rate of rejection and the incidence of graft-versus-host disease in recipients of small intestine transplantation. To determine whether a jejunal or an ileal graft is preferable with respect to intestinal morphology and function, syngeneic two-step small intestinal transplantations were performed using male Lewis rats (RTI’). They were divided into two groups according to the small intestine donor site (group 1 received 10 cm of jejunum; group 2 received 10 cm of ileum). There was no significant difference in the survival rate or weight gain between the two groups. Nearly all the hematologic findings, serum nutritional parameters, and results of liver function tests were normal for both groups. The only difference was that the serum total bile acid level was significantly higher in group 2. Fifty weeks after transplantation, the graft mucosa showed normal architecture, with adaptive hyperplasia of villi and crypt noted through histological study. The villus height of group 1 was 595 ? 64 pm (control, 452 f 67 pm); that of group 2 was 732 2 53 pm (control, 217 ir 20 pm). The crypt depth of group 1 was 228 + 35 pm (control, 165 f 24 pm); that of group 2 was 320 2 19 urn (control, 102 f 19 pm). These compensatory changes were more pronounced in group 2. The authors conclude that, on the basis of long-term functional capacity, there was no significant difference between jejunal and ileal grafts, and that both segments were suitable for transplantation. However, the ileal graft was considered to be better with respect to morphological adaptation. Copyright o 1996 by WA Saunders Company
Yamada,
and Sachiyo
Suita
with SBS. Small intestinal segments can be obtained from living-related donors, which not only increases the number of available grafts but also the chances of compatibility.4 Recent reports have shown that there is no significant difference in the immunologic response between ileal and jejunal grafts,5,6 and have suggested that, in light of rejection and graft-versushost disease (GVHD), the small intestine donor site should be selected on the basis of its distinctive functional capacity. 5 Clinical cases of SIT, using effective immunosuppressants such as cyclosporine7-9 or FK-506,10J1 have been reported recently from several institutions. However, the long-term survival rate was not as good as for other organs. In addition, to overcome the immunologic problems, it is also important to select the most suitable part of the intestine to be used in transplantation. The purpose of the present study, in which we compared the long-term morphological and functional results achieved with short segment jejunal and ileal isografts, was to determine which graft would be preferable for transplantation. MATERIALS
AND METHODS
Animals and Groups INDEX WORDS: Small intestine, ileum, total bile acid, villus height.
transplantation,
jejunum,
T
HE IMPROVEMENT in total parenteral nutrition (TPN) and its modified versions, such as home parenteral nutrition, have significantly improved the quality of life of patients with short bowel syndrome (SBS). However, a number of serious complications (eg, catheter-related sepsis, metabolic imbalance, depressive reaction) are believed to be inevitable with long-term use of TPN.rW3 Therefore, small intestinal transplantation (SIT) is becoming more popular as the radical treatment for patients
From the Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Supported in part by a Grant-in-Aid for General Scientijic Research (C) and by a Grant for Pediatric Research (6C-02) both from the Ministry of Health and Welfare, Japan. Address reprint requests to Muhammad S. Rahman, MD, Department of Pediattic Surgery, Faculty of Medicine, Kyushu University, 3-I-1, Maidashi, Higashi-ku, Fukuoka 812-82, Japan. Copyright o 1996 by WB. Saunders Company 0022-346819613107-0009$03.00/0 908
Syngeneic SIT was performed using male Lewis (RTl’) rats (Charles River Co, Ltd, Yokohama, Japan), weighing 200 to 250 g. They were divided into two groups, according to the small intestine donor site. Group 1 (n = 8) received a lo-cm segment of proximal jejunum from the ligament of Treitz. Group 2 (n = 9) received a lo-cm segment of terminal ileum.
Transplantation Procedures The rats, both donor and recipient, were fasted overnight but allowed to drink water ad libitum. Orthotopic segmental SIT was performed in two steps, according to a modification of the methods first described by Monchick and Russell.12 Briefly, the donor, under sedation with ether, was anesthetized by intraperitoneal administration of pentobarbital sodium (40 mgikg body weight) with atropine sulfate (0.05 mgikg body weight). Chloral hydrate (160 mgikg body weight) was added by intravenous or intraperitoneal administration, as necessary. Under operative microscopy (Wild M715; Wild Leitz, Heerbrugg, Switzerland), a lo-cm segment of proximal jejunum, or distal ileum, from the donor was isolated on a pedicle of the aorta and portal vein. Immediately after graft harvesting, the intestinal lumen was irrigated with cold (4°C) lactated Ringer’s solution (LR) containing 1 mg/mL Amicamycin sulfate (Meiji Pharmaceutical, Tokyo, Japan); the aorta of the graft was flushed with cold LR containing 10 UlmL of heparin. The graft was preserved in cold LR until transplanted. The recipient was anesthetized in the same fashion as the donor, and an intravenous infusion of LR was administered, at a rate of 10 mL/h, via the cannulated tail JournalofPediatric
Surgery,
Vol31,
No 7 (July),
1996:
pp 908-911
SEGMENTAL
INTESTINAL
vein or the jugular vein. The intestinal isograft was revascularized immediately by performing end-to-side anastomosis of the donor aorta to the recipient infrarenal aorta, and of the donor portal vein to the recipient inferior vena cava, using 9-O nylon sutures. Both ends of the graft were exteriorized through the right abdominal wall, as a Thiry-Vella loop. The entire native small intestine, from the ligament of Treitz to 0.5 cm proximal to the ileo-cecal valve, was replaced with the graft 7 to 10 days after transplantation. The graft was placed in functional gastrointestinal continuity with two end-to-end intestinal anastomoses between the remaining native bowel and the isograft. Deaths owing to technical failure (approximately 10%) that occurred within 72 hours of operation were excluded from the study.
Follow- Up Studies Immediately after transplantation, the recipients were allowed free access to water and standard rat food. After inserting the graft into gastrointestinal continuity, the animals received water ad libitum. A normal laboratory diet was resumed 48 hours later. Evaluation of the general health status of the animals included examination of appearance, posture, and feeding habits. The weight of the animals was examined at l-week intervals.
Biochemical
909
TRANSPLANTATION
Studies
Blood samples (2 mL) were obtained from each animal 1,4, and 50 weeks after transplantation. These samples were taken from the tail vein. Ether was used for sedation. Complete blood count (CBC) (RBC, hemoglobin, hematocrit, WBC, platelet), serum nutritional parameters (total protein, albumin, cholesterol), and liver function test results (glutamic pyruvic transaminase [GPT], bilirubin, total bile acid) were studied.
Histological Examination Five rats from each group were killed 50 weeks after transplantation and used for histological examination. Biopsy specimens of the transplanted intestine were obtained and fixed in 10% formaldehyde. After being embedded in paraffin, circular samples were stained with hematoxylin and eosin and examined by light microscopy. Five samples (each about 1 cm long) of both jejunum and ileum were obtained at the time of donor operation and were used as controls for the histological study.
Statistical Analysis All data were expressed as mean 2 standard deviation. Statistical analyses were performed using the generalized Wilcoxon, Cox-Mantel, and Student’s f tests. Probability values of less than .05 were considered significant.
%
Group 2(n=9)
IOO-
I
BO-
g 2 602 s 40-
Group
zo0
20
0
Wilcoxon and Cox-Mantel tests (Fig l), there was no significant difference in the survival rates of the two groups.
60
There
were
80
120
week
differences
be-
100
no significant
tion period. There was no statistically significant difference in weight gain between the two groups. Biochemical Evaluations The RBC, WBC, serum total protein, and GPT did not differ significantly between the two groups. The results of the hematologic study are shown in Table 1; those of the biochemical study appear in Table 2. The results were normal except for the significantly higher (I’ < .05) serum total bile acid level in group 2 (Table 2, Fig 2). The increased level of bile acid was first noted 1 week posttransplantation, and was confirmed 4 and 50 weeks after transplantation. Histological Findings The mucosal villus height of the proximal jejunum and of the terminal ileum in the control study was 452 4 67 pm and 217 r 20 km, respectively (Table 3). The corresponding crypt depths were 165 2 24 km and 102 & 19 km. The mucosal villus height of the grafts in group 1 and group 2 was 595 +- 64 km and 732 ? 53 km, respectively. The extention ratios of graft villus height for the two groups (compared with controls) were 1.32 and 3.32, respectively. The corresponding mucosal crypt depths were 228 L 35 brn and 320 f 19 km. The extention ratios of crypt depth of the grafts in groups 1 and 2 (compared with controls) were 1.38 and 3.14, respectively. The extention rates of villus height and crypt depth were higher in group Table
1. Hematologic
Parameter
Hemoglobin level (g/dL)
Hematocrit
Platelet
Weight Gain All rats ate normally, remained healthy, and gained weight at a satisfactory rate throughout the observa-
40
Fig 1. Survival curve. tween the two groups.
RESULTS
Survival All but three of the rats that had transplantation had excellent results. According to the generalized
1 (x1=8)
count
Week
Group 1
1 4
12.1 T 1.6 14.9 2 0.7 14.8 2 1.7
50 1 4 50 1
(%)
(104/mm3)
4 50 NOTE. groups.
There
were
no
Findings
significant
39.9 -+ 5.4 46.5 + 1.6 45.4 + 3.3 104 2 10.2 91.6 zi 37.3 48.3
+ 16.8
differences
Group
2
12.2 c 2.0 14.6 t 0.5 16.1 CL 2.0 37.7 2 7.9 46.7 + 2.1 50.3 k 5.1 95.1 91.4 57.0 between
2 30.7 + 16.1 + 10.9 the
two
910
RAI-IMAN
Table
2. Nutritional
Parameter Albumin
level
Cholesterol
Total
Total
level
Abbreviation:
acid
level
Function
Parameters
Table
Group 1
Group 2
P Value
1 4
3.6 + 0.3 4.1 2 0.6
3.7 + 0.2 4.1 + 0.2
NS
50 1
4.0 ? 0.6 74.5 -t 14.4 77.8 84.4 0.56
-t 6.6 + 21.1 zk 0.22
NS NS NS
(mg/dL)
4 50 1
4.5 2 0.3 60.2 2 19.2 73.9 2 7.1 87.3 0.53
0.69 -t 0.37 0.54 i 0.42 14.2 c 6.8
0.51 0.55 90.7
NS NS NS
(pmol/L)
4 50 1
+ 4.6 e 0.31 r 0.13
4 50
17.9 IL 8.5 17.8 ” 13.3
101.2 73.8
zz 0.44 ‘- 63.2 A 21.8
NS c.05 <.05
25 48.7
< .05
(mg/dL)
level
Liver
Week
(g/dL)
bilirubin
bile
and
NS
Analysis
of Intestinal
Mucosa
Group 1 Villus height Control Graft (50~wk) Extension rate Crypt depth Control Graft (50-wk) Extension rate VillusCrypt ratio Control Graft
(50~wk)
Group
2
452
f 67 Pm
217
t 20 pm
595
2 64 pm 1.32
732
+ 53 pm 3.32
165 t 24 pm 228 +- 35 pm 1.38
102 * 19 pm 320 f 19 pm 3.14
2.79 2.61
2.13 2.29
NS, not significant.
2. The ratios of villus height to crypt depth of jejunum in the control group and group 1 were 2.79 and 2.61, respectively. These ratios for the control group and group 2 were 2.13 and 2.29, respectively. DISCUSSION
With respect to clinical SIT, several factors should be considered when selecting the segment and the length of donor small intestine. These are availability of the segmental graft from living-related donors (which increases the chance of compatibility), the smaller quantity of lymphoid tissue with the segmental graft (which may induce a lesser immunologic reaction), limited space in the abdominal cavity, and the capability of the segmental graft to provide the recipient’s nutritional needs. These are the principal advantages of the segmental intestinal graft over the whoIe intestinal graft. Segmental human SIT has been used in clinical practice. The jejunum, which has less lymphoid tissue and higher enzyme concentrations, has been considered the segment of choice.13 However, Revillon et al7 used a jejuno-ileal segment, and Todo et ali0 used the
~mol/L 150
3. Morphological
ET AL
0.
l$
0
:
Fig 2. Serum total bile acid level. Compared with group had a significant increase in the serum total bile acid level. level was maintained throughout the period of observation.
1, group 2 This high
entire small intestine (with or without the colon) as intestinal allografts in recent clinical transplantations. Kimura et all4 confirmed that the frequency and severity of GVHD correlated with the length of the transplanted intestine. Another advantage of segmental SIT is that lower doses of immunosuppressants are required for good graft tolerance.15 Thus, segmental SIT had many advantages and no major disadvantages. Lossing et all6 showed that GVHD and signs of rejection occurred with the same frequency in both types of intestinal segments. It also has been reported that the time of onset of rejection and GVHD was not related to the segment of transplanted small intestine.‘” Other investigators have shown no significant difference in posttransplant immunologic responses between jejunal and ileal grafts in the rat mode1.5J3 Stangel et al5 suggested that, in light of both GVHD and rejection, the selection of the portion of small intestine to be used for transplantation should be made on the basis of distinctive absorptive capacity rather than of immunologic difference. In another study, Stangel et ali7 reported that equal doses of cyclosporine did not prolong survival, nor alter the course of rejection of jejunal or ileal grafts. In a dog model, Benchimol et al6 noted no major difference between jejunal and ileal allografts with respect to GVHD or rejection. Considering all these studies, it appears that there was no significant difference in the immunologic response between jejunal and ileal grafts. Therefore, measurement of the functional and morphological capacities after transplantation are of the utmost importance in selecting the portion of intestine to be transplanted. In our study, both intestinal segments exhibited satisfactory functional performance. The eating habits of the rats were normal. Two weeks after transplantation, the bulk and consistency of their stool output were the same as those of normal rats. Regardless of the type of graft, the necessary functions of digestion and absorption were maintained. Both groups had a good survival rate and weight gain. Our results
SEGMENTAL
INTESTINAL
911
TRANSPLANTATION
showed that a transplanted segment of as short as 10 cm permitted growth and long-term survival on a normal diet. The CBC results and serum nutritional parameters were normal. There were no significant differences between the two groups. The only difference with respect to liver function was that rats with ileal grafts had a significantly higher level of serum total bile acid. This may be explained by the presence of an active transport system for bile acid in the ileum and the systemic venous drainage of the graft (partial portosystemic shunt). The diagnosis of rejection in SIT is questionable. Except for histological examination, there are few practical and effective methods for the early diagnosis of rejection. Moreover, biopsy specimens must be obtained endoscopically, and such specimens may be inadequate for diagnosis. 18,19In addition, histological changes may be best seen in submucosa and muscle tissue. However, obtaining biopsy specimens from the intestinal wall, including deeper layers, would significantly increase the risk of perforation.18 Therefore, to gain diagnostic certainty in the monitoring of allograft rejection, additional markers must be introduced along with the histological examination. The high level of serum bile acid detected in transplanted ileal grafts could act as a possible serum marker of rejection, but further experimentation is necessary.
Histologically, the grafted intestine in the present study had a normal architecture. Villus height and crypt depth were higher in the jejunum than in the ileum. However, 50 weeks after transplantation, villus height and crypt depth increased, and were higher in the ileal grafts. This indicates that the ileal mucosa has a higher capability of morphological adaptation. Our finding that the ileum possess the higher morphological adaptability is identical to that of Hanson et a1,20who studied a short bowel model in the rat. They also showed that bile, pancreatic juice, secretin, cholecystokinin, somatostatin, and gastrin were among the potential mediators of intestinal adaptive changes. However, they did not show any difference in their production between the jejunal and ileal groups. In conclusion, the similar functional performance of ileal and jejunal segments make both suitable for grafting in SIT. Morphologically, the ileal segment may be better than the jejunal segment. Further investigations are needed to determine whether the increased level of bile acid in the ileal graft can act as a serum marker of rejection when the ileum is transplanted as an allograft. ACKNOWLEDGMENT The authors are grateful remain anonymous.
to their
proofreader,
who would
like to
REFERENCES 1. Scribner BH, James JC, Christopher TG, et al: Long term total parenteral nutrition; The concept of an artificial gut. JAMA 212:457-463,197O 2. Wolfe BM, William HB, Hayashi JT, et al: Experience with home parenteral nutrition. Am J Surg 146:7-14, 1983 3. Kurkchubache AG, Smith SD, Rowe MI, et al: Catheter sepsis in short-bowel syndrome. Arch Surg 127:21-25,199Z 4. Fortner JG, Sichuk G, Litwin SD, et al: Immunological responses to an intestinal allograft with HL-A-identical donorrecipient. Transplantation 14:531-535,1972 5. Stangel MJ, Schraut WH, Moynihan HL, et al: Rejection of ileal versus jejunal allografts. Transplantation 47:424-427,1989 6. Benchimol D, Alain P, Bayer PD, et al: Jejunal versus ileal segmental allografts in the dog; Comparison of immunologic and functional results. Surgery 112:918-927,1992 7. Revillon Y, Jan D, Goulet 0, et al: Small bowel transplantation in seven children; Preservation technique. Transplantation Proc 23:2350-2351, 1991 8. Goulet 0, Revillon Y, Canioni D, et al: Two and one-halfyear follow-up after isolated cadaveric small bowel transplantation in an infant. Transplant Proc 24:1224-1225,1992 9. McAlister V, Wall W, Ghent C, et al: Successful small intestinal transplantation. Transplant Proc 24:1236-1237, 1992 10. Todo S, Tzakis A, Reyes J, et al: Small intestinal transplantation in human with or with out the colon. Transplantation 57:840848,1994 11. Hashimoto T, Zhong RZ, Bertha M, et al: Treatment with FK506 prevents rejection of rat colon allografts. Transplantation 57:1548-1554,1994
12. Monchick GJ, Russell PS: Transplantation of small bowel in the rat; Technical and immunological considerations. Surgery 70:693-701,197l 13. Pirenne J, Disilva M, Hamoir E, et al: Influence of the length of the small bowel graft on the severity of graft versus host disease. Microsurgery 11:303-308,199O 14. Kimura K, Money SR, Jaffe BM, et al: The effect of size and site of origin of intestinal grafts on rat small bowel transplantation. Surgery 101:618-622,1987 15. Kimura K, Money SR, Jaffe BM, et al: The effect of cyclosporine on varying segments of small-bowel grafts in the rat. Surgery 4:64-69,198s 16. l&sing A, Nordgren S, Cohen Z, et al: Histologic monitoring of rejection in small intestinal transplantation. Transplantation Proc 14:643-645, 1982 17. Stangel MJ, Schraut WH, Moynihan HL, et al: Effect of cyclosporine therapy in controlling the rejection of ileal versus jejunal allografts. Transplant Int 3:149-155, 1990 18. Schmid T, Oberhuber G, Korozsi G, et al: Histologic pattern of small bowel allograft rejection in the rat; Mucosal biopsies do not provide sufficient information. Gastroenterology 96:1529-1532, 1989 19. Millard PR, Dennison A, Hughes DA, et al: Morphology of intestinal allograft rejection and the inadequacy of mucosal biopsy in its recognition. Br J Exp Path01 67:687-698, 1986 20. Hanson WR, Osborne JW: Compensation by the residual intestine after intestinal resection in the rat. Gastroenterology 72:701-705,1977