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Segmental intestinal transplantation in rats with resected entire small bowel, ileocecal valve, and cecum
JOURNAL
OF SURGICAL
Segmental
KEICHU
RESEARCH
45, 349-356 (1988)
Intestinal Transplantation in Rats with Resected Entire Small Bowel, lleocecal Valve, and Cecum’12 KIMURA,
CHARLES A. LAROSA, M.D., SAMUEL R. MONEY, AND BERNARD M. JAFFP, M.D.
M.D.,
M.D.,
Department of Surgery, State University of New York Health Science Center at Brooklyn, Brooklyn New York 11203 Submitted for publication November 1, 1987 Segmental intestinal transplantation was studied in a rat model of severe short gut syndrome across major histoincompatibility barriers. Lewis (RTl’) recipient rats whose entire small bowel (approximately 80 cm), ileocecal valve, and cecum were resectedand who had no transplant, uniformly died of malabsorption on Day 9.8 + 0.4 (n = 11). Without cyclosporine, allograft recipients (n = 2), died of rejection on Days 8 and 10. Recipient animals with 20-cm jejunum and 40-cm jejunal transplants from Buffalo (RTlb) rats and treated daily with cyclosporine (5 mg/kg/day) intramusculary (Days O-28) and vitamin Bi2 (every other week) enjoyed significantly prolonged survival to Day 58.2 + 13.7, PC 0.003, n = 10, andDay 129.1 ? 7.4,P
On the other hand, patients with short bowel syndrome, particularly, those whose After massive small bowel resection, paileocecal valves were resected, have a very tients with malabsorption require treatment high mortality rate due to malabsorption. In with total parenteral nutrition. However, those patients, intestinal transplantation fatal septicemia and physiological problems may be the only long-term viable modality. occasionally arise in those patients requiring Nonetheless, even in the rat, intestinal translong-term therapy [ 1, 41. Intestinal transplantation has not been studied in such a plantation is potentially the ideal alternative severe model of short gut syndrome (in for surgical management of the short gut which the ileocecal valve was resected). The syndrome [ 131. High doses of immunosupaim of this study was to evaluate the effecpressive agents, longer segments of intestinal tiveness of segmental jejunal allografts in a grafts, and the use of ileum (compared to model of severeshort gut syndrome in which jejunum) are all risk factors for successful the entire small bowel, the ileocecal valve, intestinal allotransplantation [7,9]. We have recently demonstrated that short segment and the cecum are resected. intestinal grafts can support the nutritional MATERIALS AND METHODS state of enterectomized rats [8]. Adult male Lewis inbred (RTl’) and Buffalo inbred (RT 1b, rats weighing 180 to 3 11 g ’ Supported in part by Foundation of Surgical Pduca- (Harlan Sprague-Dawley) were used. Lewis tion and Investigation. ’ Presented at the Annual Meeting of the Association rats were subjected to resection of the entire small bowel (approximately 80 cm) plus the for Academic Surgery, Orlando, FL, November l-4, 1987. ileocecal valve and cecum. Segmental intesINTRODUCTION
349
OO22-4804188%1SO Copyright 8 1988 by Academic Press,Inc. All rights ofreproduction in any form reserved.
350
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988
tinal grafts were taken from Buffalo rats with with one layer ( 12- 16 sutures) of interrupted the vascular pedicle attached. Orthotopic in- 6-O sutures in end-to-end fashion. testinal transplantation was performed using The donor operation. In preparation of the microsurgical technique. Four groups of ani- segmental intestinal grafts, the attached vasmals were studied. Group 1 (control group) cular pedicles, including the portal vein and consisted of 11 rats who had no transplant. the segment of aorta containing the superior Rats in groups 2(n = 10) and 3(n = 10) had mesenteric artery, were separated freely from 20-cm jejunum and 40-cm jejunal trans- the colon and pancreas. After systemic hepaplants, respectively, and received cyclospor- rinization via the inferior vena cava (150 ine, 5 mg/kg/day intramuscularly, daily units) and intraluminal irrigation of the infrom Day 0 to 28 as well as vitamin B12every testinal grafts with cold (4°C) normal saline other week. Group 4 animals (n = 2) had solution in situ, the segmental intestinal 40-cm jejunal transplants but were not given grafts with the portal veins and segments of cyclosporine. aorta were harvested. The graft vascular pedSurgical procedures (Fig. 1). Animals were icles were not perfused. Grafts were preanesthetized by pentobarbital, 35 mg/kg, in- served in cold (4°C) normal saline solution traperitoneally which was supplemented until the recipient animals were prepared. The recipient operation. Both the inferior with ether inhalation as needed. The abdominal cavity of each rat was entered by midline vena cava and abdominal aorta just distal to incision. In short gut control animals, the the left renal vein were exposed. Under a disentire jejunum, ileum, ileocecal valve, and secting microscope, two vascular anastomocecum were resected from the ligament of seswere performed between the donor portal Treitz to the proximal part of colon. Duo- vein and the recipient inferior vena cava and denocolonic anastomoses were performed between the donor aortic segment (with the superior mesenteric artery) and the recipient abdominal aorta, both in end-to-side fashion. Following the revascularization of the intestinal grafts, the recipients’ native entire jejunum, ileum, ileocecal valve, and cecum were resected.The segmental intestinal grafts were orthotopically placed between the recipient duodenal stump and the colonic margin in isoperistaltic fashion. Functional intestinal continuity was restored by two end-to-end bowel anastomosis in which the recipient duodeno-graft proximal jejunal anastomosesand graft distal jejuno-recipient colonic anastomoses were performed with 12- 16 interrupted 6-O silk sutures. Gentamycin (4 mg) and 4 cc normal saline solution were administered into the peritoneal cavity of each animal before closing. Animals were TRANSPLANTATION kept under an infrared lamp until they awoke and were returned to regular individcolon from ual cages. Pre- and postoperatively, animals were permitted free access to standard rat chow and water. Cyclosporine regimen. Cyclosporin A (Sandoz, East Hanover, NJ) was dissolved in FIG. 1. Surgical procedures: entire small bowel and olive oil at 5 mg/ml. In groups 2 and 3, the cecum resection.
KIMURA
351
ET AL.: SMALL BOWEL TRANSPLANTATION
diarrhea, despite taking standard rat chow and water. By Day 7 after operation, they lost 38.8 f 0.7% of their initial mean preoperative body weight (Fig. 2). At necropsy, bowel anastomoses were intact. There were no abdominal abscessesnor evidence of intestinal obstruction. No-cyclosporine animals. Animals in group 4 (transplantation but no cyclosporine) died of rejection on Days 8 and 10. At necropsy, the vascular and bowel anastomoses were intact and patent. The bowel walls were thin and had hemorrhagic lesions characteristic of rejected bowel. Histologically, there was destruction of the mucosa and a marked cellular infiltrate [ 161. Segmental intestinal transplants. Lewis recipient rats who had received 20- and 40-cm jejunal grafts from Buffalo rats survived significantly longer (58.2 + 13.7 days, P < 0.003, and 129.1 + 7.4 days, P < 0.001, respectively) than the no-transplant animals (9.8 f 0.4 days). Of the 10 rats who received 20-cm jejunal transplants, 7 died of malabsorption between Days 14 and 58. Clinically, they were taking both rat chow and water yet had diarrhea. At the time of necropsy, intestinal grafts retained normal appearance and there was no evidence of abscessor obstruction. Histologically, the architecture of the RESULTS intestine was relatively well preserved. Two Short gut control animals. All rats (n = 11) of the remaining 3 rats died on Days 93 and whose entire jejunum, ileum, ileocecal valve, 100 due to rejection as confirmed by histoland cecum were resected died of malabsorp- ogy. Only one rat survived for 150 days after tion between Days 8 and 12 (Table 1). These transplantation. In contrast, rats (n = 10) rats progressively lost body weight and had who had 40-cm jejunal grafts progressively recipient animals were given cyclosporine, 5 mg/kg/day intramuscularly, daily from Day 0 to Day 28. In these two groups, vitamin B12 (1 pg) was also administered intramuscularly every other week to prevent mortality from anemia due to vitamin B12deficiency [8]. Survival and daily body weight were observed. Postmortem examination was performed on all animals that died. Three rats that died within 72 hr of operation were considered as technical failures and were excluded from the study. Rats that survived at least 150 days after transplantation were sacrificed by an overdose of intraperitoneal pentobarbital injection and postmortem examination was performed. Morphological study of intestinal grafts. Intestinal grafts were preserved in 10% formalin solution. The circumference of the grafts was measured 2 cm proximal from the distal bowel anastomotic line and the data were recorded. Subsequently, all specimens were studied using 4 pm hematoxylin and eosin-stained sections and light microscopy. Statistical analysis. All values were expressed as means + SEM. Analysis of variance and Student’s t test were utilized to perform statistical analysis. P values of < 0.05 were considered as statistically significant.
TABLE 1 SURVIVALOFRATSWITHRESECTEDENTIRE
SMALLBOWEL,ILEOCECALVALVE,ANDCE~UM Survival (days)
No-transplant control Group 1 (n = 11) Allotransplant with CSA Group 2 (n = 10) 20-cm jejunum Group 3 (n = 10) 40-cm jejunum Allotransplant without CSA Group 4 (n = 2) 40-cm jejunum
8, 8, 9, 9, 10, 10, 10, 10, 11, 11, 12 14, 23, 24, 36, 36, 48, 58, 93, 100, > 150 89,94, 118, 122, 127, 141,>150,>150, >150,>150 8, 10
* P < 0.003, **P -z 0.001 compared to the no-transplant control (analysis of variance).
Mean k SEM (9.8 k 0.4) (58.2 + 13.7)* (129.1 It 7.4)** (9.0 f
1.0)
352
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988 130 N^ 40cm jejunal
transplant
20cm jejunal
60 501
transplant
4 A4, No transplant 123456789 Time after
operation
(weeks)
FIG. 2. The change in body weight with time. Short gut control Lewis rats whose entire small bowel, ileocecal valve, and cecum were resected, had no transplant (A). The recipients with severe short gut syndrome who had 20-cm (0) and 40-cm (0) jejunal transplants from Buffalo rats progressively gained body weight after the initial loss.
gained weight after the initial loss. All rats survived until at least Day 89, despite the cessation of cyclosporine administration on Day 28. Six of the 10 rats in group 3 died of rejection between Day 89 and day 141 (Table 2). Histologically, mucosal destruction and submucosal fibrosis were seen. Four rats survived for 150 days after transplantation. In these 4 rats, the diameters of the intestinal grafts 2 cm from the distal end of the grafts were compared to those of the initial grafts. At the time of transplantation (Day 0), the mean diameter of the intestinal grafts was 4.8 + 0.3 mm (mean + SEM). This did not differ from the diameter of the jejunum in normal control rats (4.8 + 0.2 mm, n = 8). However, at the time of sacrifice (150 days), the mean diameter of grafts was significantly greater, 12.8 + 1.1 mm (P < 0.001). Macroscopically (Fig. 3), the intestinal lumen of each graft was widely patent and the graft walls were slightly thickened. Microscopi-
tally (Fig. 4), tall villi and thick muscular layers were seen; some portions of the mucosal layer were destroyed by rejection. The relationship between the circumference of the intestinal grafts and the time after transplantation was plotted in Fig. 5. Presumably because postmortem pathological changes influenced the measured circumference of intestinal grafts, there was no statistically TABLE 2 CAUSES OF DEATH
Group 1. No transplant
II. 20-cmjejunum III. 4Ckm jejunum IV. Without CSA
Time of mortality Days 8-12
Days 14-58 Days93-100 715odays Days 89- I4 I 7150 days Days 8-10
Causesof death 1l/l 1 Malabsorption 7/IO Malabsorption 2/ 10 Rejection
l/IO Survivor 6110 Rejection 4/10 Survivors 212 Rejection
KIMURA
ET AL.: SMALL BOWEL TRANSPLANTATION
353
We propose this fatal model of severe short bowel syndrome for studies of the efficacy of intestinal transplantation, rather than the preparation in which only the small intestine is resected. In terms of absorptive function, the entire small bowel could be transplanted to patients with short gut syndrome. However, more practical solutions should be considered. The length and donor site of the intestinal graft harvested are quite important to successful intestinal allotransplantation. At the present time, we prefer segmental jejunal grafts. First, as the small bowel graft itself contains abundant lymphatic tissue (unlike other solid organ transplants), graft-versus-host disease is also a problem [ 141. Numerous FIG. 3. Macroscopic specimen. A normal control segtechniques to prevent graft-versus-host disment of intestine (upper) is compared to an intestinal easehave been reported [3, 10, 1I]. Decreasallograft harvested at Day 150 after transplantation (lower). ing the length of intestinal grafts or taking jejunum rather than ileum significantly reduced the mortality from graft-versus-host significant positive relationship docudisease [2, 71. Second, longer segment intesmented. tinal grafts require larger doses of cyclosporine to prevent rejection [9]. High-dose cycloDISCUSSION sporine regimens increase mortality due to Patients with short bowel syndrome, par- infection and nephrotoxicity. Thus, longer ticularly those whose ileocecal valves are re- segments, ileal grafts, and high-dose cyclosected, have high morbidity and mortality sporine regimens are avoidable risk factors. rates due to malabsorption [ 1, 41. Intestinal We previously demonstrated that short segtransplantation is the most logical surgical ment intestinal grafts permit progressive alternative. Nonetheless, even in experimen- growth of enterectomized rats [8]. The only tal animal models, intestinal transplantation disadvantage of jejunal grafts noted to date is has never been investigated in recipients their lack of ability to absorb vitamin Br2 and whose entire small bowels including the ileo- bile acids. This can easily be overcome by cecal valve were resected. Rats tolerate small regular administration of vitamin B12. bowel resection very well. Until 80% of the The protocol utilized cyclosporine treatintestine is resected, rats suffer no added ment for 28 days, after which the drug was mortality [5], presumably because the re- discontinued. It is important to emphasize maining intestine has compensatory adap- the total absence of rejection during the tive function. However, resection of 99% of treatment period. Indeed, all animals that the bowel from the ligament of Treitz to 5 did not succumb to malabsorption survived mm proximal to the ileocecal valve allowed for >60 days after cessation of therapy and for a mean 55.5 * 10.4 days survival, al- almost 40% survived for >4 months without though eventually there was 100% mortality immunosuppressive therapy. by Day 103 [8]. In the current study, animals In this study, segmental jejunal grafts have whose entire small bowels including the ileo- been investigated using a model of severe cecal valve and cecum were resected suffered short bowel syndrome. Animals that underfrom a greater degree of malabsorption. They went resection of the entire small bowel (apsurvived only an average of 9.8 f 0.4 days. proximately 80 cm), ileocecal valve, and
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JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988
FIG. 4A. Microscopic specimen (H-E Stain) of an intestinal allografi harvested at 150 days after
tr,ansplantation. Note the changes of chronic rejection.
cecurn all died of malabsorption by Day 12, havin lg lost 40% of their body weight in a week,, Segmentsequivalent to one-quarter of the le!ngth of the small bowel (20-cm jejunal grafts) resulted in 70% mortality from malab-
sorption, death occurring between Days 14 and 58. On the other hand, segmentsequi valent to half the length of the small intest ine (40~cm jejunal grafts) permitted progresswe growth with no mortality in severe short
KIMURA
ET AL.: SMALL BOWEL TRANSPLANTATION
355
FIG. 4B. Microscopic specimen of normal small intestine for comparison with Fig. 4A.
bowel syndrome recipients until Day 89 after transplantation, despite the cessation of cyclosporine injection at Day 28. These data
Time after
demonstrate that transplantation must inelude at least half the normal length of the intestine in order to permit growth of recipi-
transplantation
(days)
FIG. 5. The circumference of intestinal grafts plotted versus time in days after transplantation. l ,20-cm jejunal grafts (Y = 0.1 IX + 18.8). 0,40-cm jejunal grafts (Y = 0.1X + 12). The coefficients of correlation were not statistically significantly different.
356
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988
ents with such severe short gut syndrome (particularly those who had resection of the ileocecal valve). In experimental transplantation using murine models of heart and kidney ahografting, daily administration of 5- 10 mg/kg of cyclosporine has been shown to prevent rejection, sometimes indefinitely [ 151.Cyclosporine at 5 mg/kg intramusculary provided therapeutic serum levels of the peptide [ 171. We believe that the cyclosporine regimen of 5 mg/kg/day (Days O-28) is reasonably acceptable in rat models. However, histological rejection was seenbeyond Day 89. Continuous regimens might be required to achieve permanent acceptance of intestinal grafts in this strain combination (Buffalo to Lewis). Indeed, several investigators [6, 10, 121 have reported that high-dose cyclosporine regimens (15-20 mg/kg/day) prevent rejection indefinitely in intestinal transplantation. However, if segmental jejunal grafts are transplanted, large daily cyclosporine doses should not be required to prevent rejection. After massive small bowel resection, the remaining bowel has the ability of intestinal adaptation. Such intestinal compensation has been studied morphologically and functionally. One of the morphological parameters is the circumference of the intestine. At Day 150 after transplantation, intestinal allografts showed significant hypertrophic changes. Further studies are necessary to confirm this preliminary result. We conclude that using segmental jejunal grafts with a relatively low-dose short-term cyclosporine regimen, intestinal transplantation is an effective surgical modality for severe short bowel syndrome in the rat. REFERENCES I. Cooper, A., Floyd, T. F., Ross, A. J., III, Bishop, H. C., Templeton, J. M., Jr., and Ziegler, M. Z. Morbidity and mortality of short-bowel syndrome acquired in infancy: An update. J. Pediatr. Surg. 19: 711, 1984. 2. Deltz, E., Mtlller-Hermelink, H. K., Ulrichs, K., Thiede, A., and Mtiller-Ruchholtz, W. Develop ment of graft versus host reaction in various target
organs after small intestine transplantation. Transplant. Proc. 13: 1215, 1981.
3. Deltz, E., Uhichs, K., Schack, T., Friedrichs, B., Muller-Rucholtz, W., Muller-Hermelink, H. K., and Thide, A. Graft-versus-host reaction in small bowel transplantation and possibilities for its circumvention. Amer. J. Surg. 151: 379, 1986. 4. Grosfeld, J. L., Rescorla, F. J., and West, K. W. Short bowel syndrome in infancy and childhood. Analysis of survival in 60 patients. Amer. J Surg. 151: 41, 1986.
5. Hanson, W. R., Osborne, J. W., and Sharp, J. G. Compensation by the residual intestine after intestinal resection in the rat. I. Influence of amount of tissue removed. Gastroenterology 720: 692, 1977. 6. Harmel, R. P., and Stanley, M. Improved survival after allogenic small intestinal transplantation in the rat using cyclosporine immunosuppression. J. Pediatr. Surg. 21: 214, 1986.
7. Kimura, K., Money, S. R., and Jaffe, B. M. The effectsof size and site of origin of intestinal grafts on small-bowel transplantation in the rat. Surgery 101: 618, 1987. 8. Kimura, K., Money, S. R., and Jaffe, B. M. Short segment orthotopic intestinal isografts and allografts in enterectomized rats. Transplantation 44: 579, 1987. 9. Kimura, K., Money, S. R., and Jaffe, B. M. The effects of cyclosporine on varying segments of small bowel grafts in the rat. Surgery 104: 64, 1988. 10. Kirkman, R. L., Lear, P. A., Madara, J. L., and Tilney, N. L. Small intestine transplantation in the rat-Immunology and function. Surgery 96: 280, 1984. 11 Lee, K. K. W., and Schraut, W. H. In vitro allograft irradiation prevents graft-versus-host disease in small-bowel transplantation. J. Surg. Res. 38: 364, 1985. 12. Lee, K. K. W., and Schraut, W. H. Structure and function of orthotopic small bowel allogratts in rats treated with cyclosporine. Amer. J. Surg. 151: 55, 1986. 13. Mitchell, A., Watkins, R. M., and Collin, J. Surgical treatment of the short bowel syndrome. Brit. J. Surg. 71: 329, 1984.
14. Monchick, G. J., and Russel, P. S. Transplantation of small bowel in the rat. Technical and immunological consideration. Surgery 70: 693, 1971. 15. Morris, R. J. Cyclosporin A. Transplantation 32: 349, 1981. 16. Rosemurgy, A. S., and Schraut, W. H. Small bowel allografts. Sequence of histologic changes in acute and chronic rejection. Amer. J. Surg. 151: 470, 1986. 17. Wasscf, R., Cohen, Z., and Langer, B. Pharmacokinetic profiles of cyclosporine in the rats. Influence of route of administration and dosage. Transplantation 40: 489, 1985.
OF SURGICAL
Segmental
KEICHU
RESEARCH
45, 349-356 (1988)
Intestinal Transplantation in Rats with Resected Entire Small Bowel, lleocecal Valve, and Cecum’12 KIMURA,
CHARLES A. LAROSA, M.D., SAMUEL R. MONEY, AND BERNARD M. JAFFP, M.D.
M.D.,
M.D.,
Department of Surgery, State University of New York Health Science Center at Brooklyn, Brooklyn New York 11203 Submitted for publication November 1, 1987 Segmental intestinal transplantation was studied in a rat model of severe short gut syndrome across major histoincompatibility barriers. Lewis (RTl’) recipient rats whose entire small bowel (approximately 80 cm), ileocecal valve, and cecum were resectedand who had no transplant, uniformly died of malabsorption on Day 9.8 + 0.4 (n = 11). Without cyclosporine, allograft recipients (n = 2), died of rejection on Days 8 and 10. Recipient animals with 20-cm jejunum and 40-cm jejunal transplants from Buffalo (RTlb) rats and treated daily with cyclosporine (5 mg/kg/day) intramusculary (Days O-28) and vitamin Bi2 (every other week) enjoyed significantly prolonged survival to Day 58.2 + 13.7, PC 0.003, n = 10, andDay 129.1 ? 7.4,P
On the other hand, patients with short bowel syndrome, particularly, those whose After massive small bowel resection, paileocecal valves were resected, have a very tients with malabsorption require treatment high mortality rate due to malabsorption. In with total parenteral nutrition. However, those patients, intestinal transplantation fatal septicemia and physiological problems may be the only long-term viable modality. occasionally arise in those patients requiring Nonetheless, even in the rat, intestinal translong-term therapy [ 1, 41. Intestinal transplantation has not been studied in such a plantation is potentially the ideal alternative severe model of short gut syndrome (in for surgical management of the short gut which the ileocecal valve was resected). The syndrome [ 131. High doses of immunosupaim of this study was to evaluate the effecpressive agents, longer segments of intestinal tiveness of segmental jejunal allografts in a grafts, and the use of ileum (compared to model of severeshort gut syndrome in which jejunum) are all risk factors for successful the entire small bowel, the ileocecal valve, intestinal allotransplantation [7,9]. We have recently demonstrated that short segment and the cecum are resected. intestinal grafts can support the nutritional MATERIALS AND METHODS state of enterectomized rats [8]. Adult male Lewis inbred (RTl’) and Buffalo inbred (RT 1b, rats weighing 180 to 3 11 g ’ Supported in part by Foundation of Surgical Pduca- (Harlan Sprague-Dawley) were used. Lewis tion and Investigation. ’ Presented at the Annual Meeting of the Association rats were subjected to resection of the entire small bowel (approximately 80 cm) plus the for Academic Surgery, Orlando, FL, November l-4, 1987. ileocecal valve and cecum. Segmental intesINTRODUCTION
349
OO22-4804188%1SO Copyright 8 1988 by Academic Press,Inc. All rights ofreproduction in any form reserved.
350
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988
tinal grafts were taken from Buffalo rats with with one layer ( 12- 16 sutures) of interrupted the vascular pedicle attached. Orthotopic in- 6-O sutures in end-to-end fashion. testinal transplantation was performed using The donor operation. In preparation of the microsurgical technique. Four groups of ani- segmental intestinal grafts, the attached vasmals were studied. Group 1 (control group) cular pedicles, including the portal vein and consisted of 11 rats who had no transplant. the segment of aorta containing the superior Rats in groups 2(n = 10) and 3(n = 10) had mesenteric artery, were separated freely from 20-cm jejunum and 40-cm jejunal trans- the colon and pancreas. After systemic hepaplants, respectively, and received cyclospor- rinization via the inferior vena cava (150 ine, 5 mg/kg/day intramuscularly, daily units) and intraluminal irrigation of the infrom Day 0 to 28 as well as vitamin B12every testinal grafts with cold (4°C) normal saline other week. Group 4 animals (n = 2) had solution in situ, the segmental intestinal 40-cm jejunal transplants but were not given grafts with the portal veins and segments of cyclosporine. aorta were harvested. The graft vascular pedSurgical procedures (Fig. 1). Animals were icles were not perfused. Grafts were preanesthetized by pentobarbital, 35 mg/kg, in- served in cold (4°C) normal saline solution traperitoneally which was supplemented until the recipient animals were prepared. The recipient operation. Both the inferior with ether inhalation as needed. The abdominal cavity of each rat was entered by midline vena cava and abdominal aorta just distal to incision. In short gut control animals, the the left renal vein were exposed. Under a disentire jejunum, ileum, ileocecal valve, and secting microscope, two vascular anastomocecum were resected from the ligament of seswere performed between the donor portal Treitz to the proximal part of colon. Duo- vein and the recipient inferior vena cava and denocolonic anastomoses were performed between the donor aortic segment (with the superior mesenteric artery) and the recipient abdominal aorta, both in end-to-side fashion. Following the revascularization of the intestinal grafts, the recipients’ native entire jejunum, ileum, ileocecal valve, and cecum were resected.The segmental intestinal grafts were orthotopically placed between the recipient duodenal stump and the colonic margin in isoperistaltic fashion. Functional intestinal continuity was restored by two end-to-end bowel anastomosis in which the recipient duodeno-graft proximal jejunal anastomosesand graft distal jejuno-recipient colonic anastomoses were performed with 12- 16 interrupted 6-O silk sutures. Gentamycin (4 mg) and 4 cc normal saline solution were administered into the peritoneal cavity of each animal before closing. Animals were TRANSPLANTATION kept under an infrared lamp until they awoke and were returned to regular individcolon from ual cages. Pre- and postoperatively, animals were permitted free access to standard rat chow and water. Cyclosporine regimen. Cyclosporin A (Sandoz, East Hanover, NJ) was dissolved in FIG. 1. Surgical procedures: entire small bowel and olive oil at 5 mg/ml. In groups 2 and 3, the cecum resection.
KIMURA
351
ET AL.: SMALL BOWEL TRANSPLANTATION
diarrhea, despite taking standard rat chow and water. By Day 7 after operation, they lost 38.8 f 0.7% of their initial mean preoperative body weight (Fig. 2). At necropsy, bowel anastomoses were intact. There were no abdominal abscessesnor evidence of intestinal obstruction. No-cyclosporine animals. Animals in group 4 (transplantation but no cyclosporine) died of rejection on Days 8 and 10. At necropsy, the vascular and bowel anastomoses were intact and patent. The bowel walls were thin and had hemorrhagic lesions characteristic of rejected bowel. Histologically, there was destruction of the mucosa and a marked cellular infiltrate [ 161. Segmental intestinal transplants. Lewis recipient rats who had received 20- and 40-cm jejunal grafts from Buffalo rats survived significantly longer (58.2 + 13.7 days, P < 0.003, and 129.1 + 7.4 days, P < 0.001, respectively) than the no-transplant animals (9.8 f 0.4 days). Of the 10 rats who received 20-cm jejunal transplants, 7 died of malabsorption between Days 14 and 58. Clinically, they were taking both rat chow and water yet had diarrhea. At the time of necropsy, intestinal grafts retained normal appearance and there was no evidence of abscessor obstruction. Histologically, the architecture of the RESULTS intestine was relatively well preserved. Two Short gut control animals. All rats (n = 11) of the remaining 3 rats died on Days 93 and whose entire jejunum, ileum, ileocecal valve, 100 due to rejection as confirmed by histoland cecum were resected died of malabsorp- ogy. Only one rat survived for 150 days after tion between Days 8 and 12 (Table 1). These transplantation. In contrast, rats (n = 10) rats progressively lost body weight and had who had 40-cm jejunal grafts progressively recipient animals were given cyclosporine, 5 mg/kg/day intramuscularly, daily from Day 0 to Day 28. In these two groups, vitamin B12 (1 pg) was also administered intramuscularly every other week to prevent mortality from anemia due to vitamin B12deficiency [8]. Survival and daily body weight were observed. Postmortem examination was performed on all animals that died. Three rats that died within 72 hr of operation were considered as technical failures and were excluded from the study. Rats that survived at least 150 days after transplantation were sacrificed by an overdose of intraperitoneal pentobarbital injection and postmortem examination was performed. Morphological study of intestinal grafts. Intestinal grafts were preserved in 10% formalin solution. The circumference of the grafts was measured 2 cm proximal from the distal bowel anastomotic line and the data were recorded. Subsequently, all specimens were studied using 4 pm hematoxylin and eosin-stained sections and light microscopy. Statistical analysis. All values were expressed as means + SEM. Analysis of variance and Student’s t test were utilized to perform statistical analysis. P values of < 0.05 were considered as statistically significant.
TABLE 1 SURVIVALOFRATSWITHRESECTEDENTIRE
SMALLBOWEL,ILEOCECALVALVE,ANDCE~UM Survival (days)
No-transplant control Group 1 (n = 11) Allotransplant with CSA Group 2 (n = 10) 20-cm jejunum Group 3 (n = 10) 40-cm jejunum Allotransplant without CSA Group 4 (n = 2) 40-cm jejunum
8, 8, 9, 9, 10, 10, 10, 10, 11, 11, 12 14, 23, 24, 36, 36, 48, 58, 93, 100, > 150 89,94, 118, 122, 127, 141,>150,>150, >150,>150 8, 10
* P < 0.003, **P -z 0.001 compared to the no-transplant control (analysis of variance).
Mean k SEM (9.8 k 0.4) (58.2 + 13.7)* (129.1 It 7.4)** (9.0 f
1.0)
352
JOURNAL OF SURGICAL RESEARCH: VOL. 45, NO. 4, OCTOBER 1988 130 N^ 40cm jejunal
transplant
20cm jejunal
60 501
transplant
4 A4, No transplant 123456789 Time after
operation
(weeks)
FIG. 2. The change in body weight with time. Short gut control Lewis rats whose entire small bowel, ileocecal valve, and cecum were resected, had no transplant (A). The recipients with severe short gut syndrome who had 20-cm (0) and 40-cm (0) jejunal transplants from Buffalo rats progressively gained body weight after the initial loss.
gained weight after the initial loss. All rats survived until at least Day 89, despite the cessation of cyclosporine administration on Day 28. Six of the 10 rats in group 3 died of rejection between Day 89 and day 141 (Table 2). Histologically, mucosal destruction and submucosal fibrosis were seen. Four rats survived for 150 days after transplantation. In these 4 rats, the diameters of the intestinal grafts 2 cm from the distal end of the grafts were compared to those of the initial grafts. At the time of transplantation (Day 0), the mean diameter of the intestinal grafts was 4.8 + 0.3 mm (mean + SEM). This did not differ from the diameter of the jejunum in normal control rats (4.8 + 0.2 mm, n = 8). However, at the time of sacrifice (150 days), the mean diameter of grafts was significantly greater, 12.8 + 1.1 mm (P < 0.001). Macroscopically (Fig. 3), the intestinal lumen of each graft was widely patent and the graft walls were slightly thickened. Microscopi-
tally (Fig. 4), tall villi and thick muscular layers were seen; some portions of the mucosal layer were destroyed by rejection. The relationship between the circumference of the intestinal grafts and the time after transplantation was plotted in Fig. 5. Presumably because postmortem pathological changes influenced the measured circumference of intestinal grafts, there was no statistically TABLE 2 CAUSES OF DEATH
Group 1. No transplant
II. 20-cmjejunum III. 4Ckm jejunum IV. Without CSA
Time of mortality Days 8-12
Days 14-58 Days93-100 715odays Days 89- I4 I 7150 days Days 8-10
Causesof death 1l/l 1 Malabsorption 7/IO Malabsorption 2/ 10 Rejection
l/IO Survivor 6110 Rejection 4/10 Survivors 212 Rejection
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We propose this fatal model of severe short bowel syndrome for studies of the efficacy of intestinal transplantation, rather than the preparation in which only the small intestine is resected. In terms of absorptive function, the entire small bowel could be transplanted to patients with short gut syndrome. However, more practical solutions should be considered. The length and donor site of the intestinal graft harvested are quite important to successful intestinal allotransplantation. At the present time, we prefer segmental jejunal grafts. First, as the small bowel graft itself contains abundant lymphatic tissue (unlike other solid organ transplants), graft-versus-host disease is also a problem [ 141. Numerous FIG. 3. Macroscopic specimen. A normal control segtechniques to prevent graft-versus-host disment of intestine (upper) is compared to an intestinal easehave been reported [3, 10, 1I]. Decreasallograft harvested at Day 150 after transplantation (lower). ing the length of intestinal grafts or taking jejunum rather than ileum significantly reduced the mortality from graft-versus-host significant positive relationship docudisease [2, 71. Second, longer segment intesmented. tinal grafts require larger doses of cyclosporine to prevent rejection [9]. High-dose cycloDISCUSSION sporine regimens increase mortality due to Patients with short bowel syndrome, par- infection and nephrotoxicity. Thus, longer ticularly those whose ileocecal valves are re- segments, ileal grafts, and high-dose cyclosected, have high morbidity and mortality sporine regimens are avoidable risk factors. rates due to malabsorption [ 1, 41. Intestinal We previously demonstrated that short segtransplantation is the most logical surgical ment intestinal grafts permit progressive alternative. Nonetheless, even in experimen- growth of enterectomized rats [8]. The only tal animal models, intestinal transplantation disadvantage of jejunal grafts noted to date is has never been investigated in recipients their lack of ability to absorb vitamin Br2 and whose entire small bowels including the ileo- bile acids. This can easily be overcome by cecal valve were resected. Rats tolerate small regular administration of vitamin B12. bowel resection very well. Until 80% of the The protocol utilized cyclosporine treatintestine is resected, rats suffer no added ment for 28 days, after which the drug was mortality [5], presumably because the re- discontinued. It is important to emphasize maining intestine has compensatory adap- the total absence of rejection during the tive function. However, resection of 99% of treatment period. Indeed, all animals that the bowel from the ligament of Treitz to 5 did not succumb to malabsorption survived mm proximal to the ileocecal valve allowed for >60 days after cessation of therapy and for a mean 55.5 * 10.4 days survival, al- almost 40% survived for >4 months without though eventually there was 100% mortality immunosuppressive therapy. by Day 103 [8]. In the current study, animals In this study, segmental jejunal grafts have whose entire small bowels including the ileo- been investigated using a model of severe cecal valve and cecum were resected suffered short bowel syndrome. Animals that underfrom a greater degree of malabsorption. They went resection of the entire small bowel (apsurvived only an average of 9.8 f 0.4 days. proximately 80 cm), ileocecal valve, and
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FIG. 4A. Microscopic specimen (H-E Stain) of an intestinal allografi harvested at 150 days after
tr,ansplantation. Note the changes of chronic rejection.
cecurn all died of malabsorption by Day 12, havin lg lost 40% of their body weight in a week,, Segmentsequivalent to one-quarter of the le!ngth of the small bowel (20-cm jejunal grafts) resulted in 70% mortality from malab-
sorption, death occurring between Days 14 and 58. On the other hand, segmentsequi valent to half the length of the small intest ine (40~cm jejunal grafts) permitted progresswe growth with no mortality in severe short
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FIG. 4B. Microscopic specimen of normal small intestine for comparison with Fig. 4A.
bowel syndrome recipients until Day 89 after transplantation, despite the cessation of cyclosporine injection at Day 28. These data
Time after
demonstrate that transplantation must inelude at least half the normal length of the intestine in order to permit growth of recipi-
transplantation
(days)
FIG. 5. The circumference of intestinal grafts plotted versus time in days after transplantation. l ,20-cm jejunal grafts (Y = 0.1 IX + 18.8). 0,40-cm jejunal grafts (Y = 0.1X + 12). The coefficients of correlation were not statistically significantly different.
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ents with such severe short gut syndrome (particularly those who had resection of the ileocecal valve). In experimental transplantation using murine models of heart and kidney ahografting, daily administration of 5- 10 mg/kg of cyclosporine has been shown to prevent rejection, sometimes indefinitely [ 151.Cyclosporine at 5 mg/kg intramusculary provided therapeutic serum levels of the peptide [ 171. We believe that the cyclosporine regimen of 5 mg/kg/day (Days O-28) is reasonably acceptable in rat models. However, histological rejection was seenbeyond Day 89. Continuous regimens might be required to achieve permanent acceptance of intestinal grafts in this strain combination (Buffalo to Lewis). Indeed, several investigators [6, 10, 121 have reported that high-dose cyclosporine regimens (15-20 mg/kg/day) prevent rejection indefinitely in intestinal transplantation. However, if segmental jejunal grafts are transplanted, large daily cyclosporine doses should not be required to prevent rejection. After massive small bowel resection, the remaining bowel has the ability of intestinal adaptation. Such intestinal compensation has been studied morphologically and functionally. One of the morphological parameters is the circumference of the intestine. At Day 150 after transplantation, intestinal allografts showed significant hypertrophic changes. Further studies are necessary to confirm this preliminary result. We conclude that using segmental jejunal grafts with a relatively low-dose short-term cyclosporine regimen, intestinal transplantation is an effective surgical modality for severe short bowel syndrome in the rat. REFERENCES I. Cooper, A., Floyd, T. F., Ross, A. J., III, Bishop, H. C., Templeton, J. M., Jr., and Ziegler, M. Z. Morbidity and mortality of short-bowel syndrome acquired in infancy: An update. J. Pediatr. Surg. 19: 711, 1984. 2. Deltz, E., Mtlller-Hermelink, H. K., Ulrichs, K., Thiede, A., and Mtiller-Ruchholtz, W. Develop ment of graft versus host reaction in various target
organs after small intestine transplantation. Transplant. Proc. 13: 1215, 1981.
3. Deltz, E., Uhichs, K., Schack, T., Friedrichs, B., Muller-Rucholtz, W., Muller-Hermelink, H. K., and Thide, A. Graft-versus-host reaction in small bowel transplantation and possibilities for its circumvention. Amer. J. Surg. 151: 379, 1986. 4. Grosfeld, J. L., Rescorla, F. J., and West, K. W. Short bowel syndrome in infancy and childhood. Analysis of survival in 60 patients. Amer. J Surg. 151: 41, 1986.
5. Hanson, W. R., Osborne, J. W., and Sharp, J. G. Compensation by the residual intestine after intestinal resection in the rat. I. Influence of amount of tissue removed. Gastroenterology 720: 692, 1977. 6. Harmel, R. P., and Stanley, M. Improved survival after allogenic small intestinal transplantation in the rat using cyclosporine immunosuppression. J. Pediatr. Surg. 21: 214, 1986.
7. Kimura, K., Money, S. R., and Jaffe, B. M. The effectsof size and site of origin of intestinal grafts on small-bowel transplantation in the rat. Surgery 101: 618, 1987. 8. Kimura, K., Money, S. R., and Jaffe, B. M. Short segment orthotopic intestinal isografts and allografts in enterectomized rats. Transplantation 44: 579, 1987. 9. Kimura, K., Money, S. R., and Jaffe, B. M. The effects of cyclosporine on varying segments of small bowel grafts in the rat. Surgery 104: 64, 1988. 10. Kirkman, R. L., Lear, P. A., Madara, J. L., and Tilney, N. L. Small intestine transplantation in the rat-Immunology and function. Surgery 96: 280, 1984. 11 Lee, K. K. W., and Schraut, W. H. In vitro allograft irradiation prevents graft-versus-host disease in small-bowel transplantation. J. Surg. Res. 38: 364, 1985. 12. Lee, K. K. W., and Schraut, W. H. Structure and function of orthotopic small bowel allogratts in rats treated with cyclosporine. Amer. J. Surg. 151: 55, 1986. 13. Mitchell, A., Watkins, R. M., and Collin, J. Surgical treatment of the short bowel syndrome. Brit. J. Surg. 71: 329, 1984.
14. Monchick, G. J., and Russel, P. S. Transplantation of small bowel in the rat. Technical and immunological consideration. Surgery 70: 693, 1971. 15. Morris, R. J. Cyclosporin A. Transplantation 32: 349, 1981. 16. Rosemurgy, A. S., and Schraut, W. H. Small bowel allografts. Sequence of histologic changes in acute and chronic rejection. Amer. J. Surg. 151: 470, 1986. 17. Wasscf, R., Cohen, Z., and Langer, B. Pharmacokinetic profiles of cyclosporine in the rats. Influence of route of administration and dosage. Transplantation 40: 489, 1985.