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Elemental Enteral Nutrition Preserves the Mucosal Barrier and Improves the Trophism of the Villi After Small Bowel Transplantation in Piglets
Elemental Enteral Nutrition Preserves the Mucosal Barrier and Improves the Trophism of the Villi After Small Bowel Transplantation in Piglets S. Zonta, M. Doni, M. Alessiani, F. Lovisetto, J. Vigano, M. Mazzilli, T. Dominioni, M. Podetta, M. De Martino, M. Scaglione, E. Vicini, A. Bottazzi, C. Villa, P. Morbini, and P. Dionigi ABSTRACT The main goals for a successful small bowel transplantation (SBTx) are the control of acute rejection and maintenance of the mucosal barrier, which plays a key role in preventing bacterial translocation and preserving absorptive capacity. According to recent evidence that sustaining enteral nutrition (EN) as rehabilitative therapy improves the integrity of the mucosal barrier after SBTx, we studied the trophic effect of a new elemental enteral solution whose proteinic supply is represented by oligomeric-aminoacidic chains. In a swine SBTx model we studied three groups, divided by the different postoperative feeding: group 1 (n ⫽ 5): standard swine chow, group 2 (n ⫽ 5): polymeric enteral solution, group 3 (n ⫽ 5): elemental enteral solution (Peptamen, Nestlè Corp). All animals were immunosuppressed with a tacrolimus/FK778 combined oral therapy. The nutritional indices evaluated were: body weight, episodes of diarrhea, D-xylose absorption test, and histopatological and villi morphometric analysis. Three pigs died before the end of the study, two in group 1 (pneumonia and sepsis), one in group 2 (pneumonia). Mean days of diarrhea were 15, 10, and 3 in groups 1, 2, and 3, respectively (P ⬍ .05). The final/starting weight ratio was 1.08 for group 3 and 0.92 for group 2 (P ⬍ .05); the D-xylose curves showed a statistically significant difference for group 3 versus the groups 2 and 1 (P ⬍ .05), as well as for the villi height (P ⬍ .01) and width (P ⬍ .05). In conclusion, elemental enteral solution, with its basic protein supply, does not require a very complex enzymatic system to be metabolized. Thus, it may contribute to a faster recovery of the mucosal barrier and to limit the hypercatabolic state.
C
ONTROL OF ACUTE REJECTION and the maintenance of the mucosal barrier function represent pivotal issues for the outcome of the small bowel transplantation (SBTx), and their mutual balance is crucial in the postoperative management. The integrity of the mucosal barrier plays a key role to prevent the bacterial translocation and increase the absorptive capacity even though many factors could reduce its function after transplantation.1,2 Recent evidence indicates that enteral nutrition (EN) could promote the preservation of an intact intestinal epithelial barrier and may reduce the bacterial endoluminal overgrowth and translocation. In fact, the intestinal mucosa is mainly supplied via the intestinal lumen, and the preservation of an intact enteral flora stabilizes the pH environmental in the intestinal lumen and the absorbitive capacity of the enteral mucosa.3 Different EN regimens enriched with
glutamine and probiotics have been tested and resulted in the improvement of mucosal barrier healing. Few investigation are nowadays available concerning the most appropriate protein supply and whether the complexity of the amino acid chain may induce the expression of surface enzymatic complexes necessary for mucosal barrier function.
From the Departments of Surgery (S.Z., M.D., M.A., F.L., J.V., M.M., T.D., M.P., M.D.M., M.S., E.V., P.D.) and Anesthesiology (A.B.), Pathology Institute (C.V., P.M.), University of Pavia School of Medicine and Fondazione I.R.C.C.S. Policlinico San Matteo Hospital, Pavia, Italy. Address reprint requests to Sandro Zonta, MD, Chirurgia Epatopancreatica, Fondazione I.R.C.C.S. Policlinico San Matteo, P.le Golgi 2, 27100 Pavia, Italy. E-mail: [email protected]
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.048
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2024
Transplantation Proceedings, 39, 2024 –2027 (2007)
ELEMENTAL ENTERAL NUTRITION
2025 Table 1. Nutritional Indices of the Study Groups
Group Group Group 4 Group 5 Group 6 Group 7 Group 2 3
3 3 3 3 3 3 3
Villi morphometry (mean ⫾ SD)
Final/staring weight (mean ⫾ SD)
POD 8
POD 30
POD 60
Height(POD 60)/Height(POD 0)
Width(POD 60)/Width(POD 0)
15.2 ⫾ 2.5 10.4 ⫾ 1.8 1 7.3 ⫾ 1.2
0.78 ⫾ 0.04 0.92 ⫾ 0.06 2 1.08 ⫾ 0.05
3.8 ⫾ 0.9 3.7 ⫾ 1.1 3 3.7 ⫾ 1.2
4.3 ⫾ 0.9 4.8 ⫾ 1.2 4 5.2 ⫾ 1.5
4.5 ⫾ 0.5 5.4 ⫾ 1.1 5 5.9 ⫾ 1.7
0.63 ⫾ 0.04 0.72 ⫾ 0.05 6 0.87 ⫾ 0.14
0.65 ⫾ 0.11 0.70 ⫾ 0.07 7 0.85 ⫾ 0.09
Group 1 Group 2 Group 3 1
Serum albumin level (g/dL, mean ⫾ SD)
Diarrhea (day, mean ⫾ SD)
vs vs vs vs vs vs vs
group group group group group group group
2: 2: 2: 2: 2: 2: 2:
P P P P P P P
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
.013; group 2 vs group 1: P ⫽ .008. .002; group 2 vs group 1: P ⫽ .002. NS; group 2 vs group 1: P ⫽ NS. NS; group 2 vs group 1: P ⫽ NS. NS; group 2 vs group 1: P ⫽ .023. 0.05; group 2 vs group 1: P ⫽ 0.014. 0.01; group 2 vs group 1: P ⫽ 0.043.
In an SBTx model in pigs, we studied the trophic effect of a new enteral solution whose protein supply was mainly represented by oligomeric-amino acidic chains (bi-, trimeric aminoacid) (Peptamen, Nestle Corp) as enteral rehabilitative therapy; we compared the results with two control groups, the first one fed with standard chow and the other with an enteral solution whose protein supply was represented by polymeric amino acidic chain (Nutrison, Nutricia Inc). MATERIALS AND METHODS Study Design and Immunosuppression Fifteen unrelated female piglets (race Large White) underwent total SBTx under combination therapy of tacrolimus and FK 778. The SBTx surgical procedure has been described elsewhere.4 The follow-up time was 2 months. Only animals that did not develop technical complications from the intestinal transplant procedure were included in the study. Oral tacrolimus was given at an adjusted dose to maintain trough level range between 5 and 15 ng/mL. Oral FK778 was administered daily at the dose of 4 mg/kg. Tacrolimus whole blood levels were determined weekly by a microparticle immunoassay. The treated pigs were randomly assigned to the following study groups: group 1 (n ⫽ 5): standard swine chow ad libitum in the postoperative period; group 2 (n ⫽ 5): polymeric enteral solution (Nutrison, Nutricia Inc) started on 4 post operative day (POD) 4 and enteral feeding administered trough a gastrostomy tube and provided 25 kcal/kg/d; group 3 (n ⫽ 5): elemetal enteral solution (Peptamen, Nestle Corp) started on POD 4 and enteral feeding administered trough a gastrostomy tube and provided 25 kcal/kg/d.
Nutritional Indices Nutritional indices included body weight, presence of diarrhea, and serum albumin concentration (determined by Hitachi 7150 Biochemical analyzer on PODs 8, 30, 60).
D-xylose Absorption Test On POD 60, the intestinal adsorption was tested evaluating the D-xylose adsorption curve, which was compared with normal untreated animals (control group, n ⫽ 10). D-xylose was given orally at day 60 through the gastrostomy at the standard dose of 0.5 g/kg, and its serum levels were measured with a UV/VIS spectrophotomer at 0 and 30, 60, 90, 120, 150 and 180 minutes after administration. Blood samples were taken from a catheter placed in a central vein at day 60.
Histology and Morphometric Indices of Graft Mucosa Full-thickness biopsies were harvested from the graft on PODs 0, 7, 14, 21, 30, 45, and 60. The new grading system of Wu et al5 was used to quantify grades of acute cellular rejection (ACR). Histopathologic analysis was performed using hematoxylin and eosin (HE) staining. Morphometric indices of the graft mucosa including villus height and villus width were investigated on tissue cross section using a MPIAS-500, a multiple media pathological image analysis system; morphometric indices measurements are expressed as ratio between the data from biopsies on POD 60 and data from day 0, before SBTx.
Statistical Analysis Data are expressed as means ⫾ standard deviation. Means between group were compared over time with two-way analysis of variance test; proportions between study groups were compared applying the chi-square test with the Yeats corrections. Comparisons among the experimental group’s curves were performed using the MANOVA test for repeated measures. Probability values (P) of less than .05 were considered to be statistically significant.
RESULTS
None of the transplanted pigs in the group 3 died before the end of the study; severe infections were the causes of death of two pigs in the group 1 (one pneumonia and one general sepsis occurred on POD 20 and in POD 32, respectively) and one pig in group 2 (pneumonia occurred on POD 18). The overall incidence of acute graft rejection in the tissue specimens examined (biopses along the study and graft samples harvested at the autopsy) was 37.5% for group 1, 23% for group 2, and 13% for group 3 (group 3 vs group 2: P ⬍ 0.05; group 2 and group 3 vs group 1: P ⬍ .001). The nutritional indices of the study groups are shown in the Table 1. Diarrhea was observed in all transplanted piglets, with a mean duration of 15.2 days, 10.4 days, and 7.3 days, respectively, for group 1, group 2, and group 3. The differences between the study groups concerning the duration of the diarrhea, ratio between final and starting weight, and morphometric villi evaluation were statistically significant, showing an advantage for the tranplanted pigs treated in group 3. The HE-stained sections of the epithelial specimen of group 3 intestinal graft harvested in the course of the study confirmed better conservation of the villi and
2026
ZONTA, DONI, ALESSIANI ET AL
Fig 1. D-xylose absorption curve reported study groups compared with control groups.
higher trophic appearance of mucosal structures compared to those of groups 1 and 2. D-xylose absorption curves observed at POD 60 in the treated groups are reported in Fig 1 and are compared with those obtained in the control group. The multivariate analysis of the four data sets applying the MANOVA test showed significant differences along the whole curves of absorption (group 3 vs group 2: P ⫽ .02; group C vs group 3; P ⫽ .04; group 2 vs group 1: P ⫽ .001). DISCUSSION
The preservation-reperfusion injury represents the most critical event during SBTx that may affect the integrity of the mucosal barrier and promote postoperative complications. The consequences of this damage are evident both on the metabolic status of the recipients and on the immunological surveillance. Fluid hypersecretion with loss of electrolytes and proteins is the result of the breakdown of the intestinal epithelium, which is associated to the release of inflammatory mediators acting on the recruitment of the antigen-presenting cells and predisposing to acute cellular rejection. The rupture of the mucosal barrier promotes bacterial translocation, causing severe infectious episodes within a transplanted organism even weakened by hypercatabolic state. The main postoperative complications— namely pneumonia, peritonitis, intestinal leakage, sepsis, ACR, and graft-versus-host disease— have the same root causes, recognizing a unique pathogenetic mechanism that could be improved with preservation of the mucosal barrier. EN was recently approved for enhancing resistance to infection in experimental models and in clinical settings; it
exerts a protective effect to immunity and nutritional status, thus influencing the rate of complication and prognosis. The employment of polymeric EN associated with the immunonutrition such as glutamine and lactobacilli showed a slight advantage toward standard EN solution.1,2 The rationale of early EN is a faster recovery of the mucosal barrier from operative trauma and prevention of starvation leading to bacterial overgrowth, endotoxemia, and release of mediators that could delay the restoration of the barrier function. The complexity of the nutrient that constitutes the EN and the number of enzymatic reactions required for the complete absorption of the substrate may result in a limiting factor reducing the efficacy of the enteral solution. Elemental enteral solution represents a new option for warranting a complete proteinic uptake when the loss of the barrier integrity may reduce the enzymatic surface activities. Our study demonstrated that the availability of elemental nutritional substrates improved the recovery of the absorptive function, highlighting that only the basic functions are preserved after SBTx. The restoration of the absorptive activity may be influenced by the quality of the nutrient. Moreover, the prevalence of easily to metabolize substrates may promptly interrupt the hypercathabolic status.
REFERENCES 1. Schulz RJ, Dignass A, Pascher A, et al: New dietary concepts in small bowel transplantation. Transplant Proc 34:893, 2002
ELEMENTAL ENTERAL NUTRITION 2. Mueller AR, Pasher A, Schulz AR, et al: Clinical small bowel transplantation: focus on mucosal function. Transplant Proc 43: 926, 2002 3. Zhang XQ, Li JS, Li N, et al: Trophic effect of enteral rehabilitative therapy in rat small bowel transplantation. Transplant Proc 37:2351, 2005
2027 4. Alessiani M, Spada M, Merli M, et al: Different models of intestinal transplantation in pigs: technical aspects. Transplant Proc 28:2720, 1996 5. Wu T, Abu Elmagd K, Bonf J, et al: A schema for histologic grading of small intestine allograft acute rejection. Transplantation 75:1241, 2003
C
ONTROL OF ACUTE REJECTION and the maintenance of the mucosal barrier function represent pivotal issues for the outcome of the small bowel transplantation (SBTx), and their mutual balance is crucial in the postoperative management. The integrity of the mucosal barrier plays a key role to prevent the bacterial translocation and increase the absorptive capacity even though many factors could reduce its function after transplantation.1,2 Recent evidence indicates that enteral nutrition (EN) could promote the preservation of an intact intestinal epithelial barrier and may reduce the bacterial endoluminal overgrowth and translocation. In fact, the intestinal mucosa is mainly supplied via the intestinal lumen, and the preservation of an intact enteral flora stabilizes the pH environmental in the intestinal lumen and the absorbitive capacity of the enteral mucosa.3 Different EN regimens enriched with
glutamine and probiotics have been tested and resulted in the improvement of mucosal barrier healing. Few investigation are nowadays available concerning the most appropriate protein supply and whether the complexity of the amino acid chain may induce the expression of surface enzymatic complexes necessary for mucosal barrier function.
From the Departments of Surgery (S.Z., M.D., M.A., F.L., J.V., M.M., T.D., M.P., M.D.M., M.S., E.V., P.D.) and Anesthesiology (A.B.), Pathology Institute (C.V., P.M.), University of Pavia School of Medicine and Fondazione I.R.C.C.S. Policlinico San Matteo Hospital, Pavia, Italy. Address reprint requests to Sandro Zonta, MD, Chirurgia Epatopancreatica, Fondazione I.R.C.C.S. Policlinico San Matteo, P.le Golgi 2, 27100 Pavia, Italy. E-mail: [email protected]
0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.05.048
© 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2024
Transplantation Proceedings, 39, 2024 –2027 (2007)
ELEMENTAL ENTERAL NUTRITION
2025 Table 1. Nutritional Indices of the Study Groups
Group Group Group 4 Group 5 Group 6 Group 7 Group 2 3
3 3 3 3 3 3 3
Villi morphometry (mean ⫾ SD)
Final/staring weight (mean ⫾ SD)
POD 8
POD 30
POD 60
Height(POD 60)/Height(POD 0)
Width(POD 60)/Width(POD 0)
15.2 ⫾ 2.5 10.4 ⫾ 1.8 1 7.3 ⫾ 1.2
0.78 ⫾ 0.04 0.92 ⫾ 0.06 2 1.08 ⫾ 0.05
3.8 ⫾ 0.9 3.7 ⫾ 1.1 3 3.7 ⫾ 1.2
4.3 ⫾ 0.9 4.8 ⫾ 1.2 4 5.2 ⫾ 1.5
4.5 ⫾ 0.5 5.4 ⫾ 1.1 5 5.9 ⫾ 1.7
0.63 ⫾ 0.04 0.72 ⫾ 0.05 6 0.87 ⫾ 0.14
0.65 ⫾ 0.11 0.70 ⫾ 0.07 7 0.85 ⫾ 0.09
Group 1 Group 2 Group 3 1
Serum albumin level (g/dL, mean ⫾ SD)
Diarrhea (day, mean ⫾ SD)
vs vs vs vs vs vs vs
group group group group group group group
2: 2: 2: 2: 2: 2: 2:
P P P P P P P
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
.013; group 2 vs group 1: P ⫽ .008. .002; group 2 vs group 1: P ⫽ .002. NS; group 2 vs group 1: P ⫽ NS. NS; group 2 vs group 1: P ⫽ NS. NS; group 2 vs group 1: P ⫽ .023. 0.05; group 2 vs group 1: P ⫽ 0.014. 0.01; group 2 vs group 1: P ⫽ 0.043.
In an SBTx model in pigs, we studied the trophic effect of a new enteral solution whose protein supply was mainly represented by oligomeric-amino acidic chains (bi-, trimeric aminoacid) (Peptamen, Nestle Corp) as enteral rehabilitative therapy; we compared the results with two control groups, the first one fed with standard chow and the other with an enteral solution whose protein supply was represented by polymeric amino acidic chain (Nutrison, Nutricia Inc). MATERIALS AND METHODS Study Design and Immunosuppression Fifteen unrelated female piglets (race Large White) underwent total SBTx under combination therapy of tacrolimus and FK 778. The SBTx surgical procedure has been described elsewhere.4 The follow-up time was 2 months. Only animals that did not develop technical complications from the intestinal transplant procedure were included in the study. Oral tacrolimus was given at an adjusted dose to maintain trough level range between 5 and 15 ng/mL. Oral FK778 was administered daily at the dose of 4 mg/kg. Tacrolimus whole blood levels were determined weekly by a microparticle immunoassay. The treated pigs were randomly assigned to the following study groups: group 1 (n ⫽ 5): standard swine chow ad libitum in the postoperative period; group 2 (n ⫽ 5): polymeric enteral solution (Nutrison, Nutricia Inc) started on 4 post operative day (POD) 4 and enteral feeding administered trough a gastrostomy tube and provided 25 kcal/kg/d; group 3 (n ⫽ 5): elemetal enteral solution (Peptamen, Nestle Corp) started on POD 4 and enteral feeding administered trough a gastrostomy tube and provided 25 kcal/kg/d.
Nutritional Indices Nutritional indices included body weight, presence of diarrhea, and serum albumin concentration (determined by Hitachi 7150 Biochemical analyzer on PODs 8, 30, 60).
D-xylose Absorption Test On POD 60, the intestinal adsorption was tested evaluating the D-xylose adsorption curve, which was compared with normal untreated animals (control group, n ⫽ 10). D-xylose was given orally at day 60 through the gastrostomy at the standard dose of 0.5 g/kg, and its serum levels were measured with a UV/VIS spectrophotomer at 0 and 30, 60, 90, 120, 150 and 180 minutes after administration. Blood samples were taken from a catheter placed in a central vein at day 60.
Histology and Morphometric Indices of Graft Mucosa Full-thickness biopsies were harvested from the graft on PODs 0, 7, 14, 21, 30, 45, and 60. The new grading system of Wu et al5 was used to quantify grades of acute cellular rejection (ACR). Histopathologic analysis was performed using hematoxylin and eosin (HE) staining. Morphometric indices of the graft mucosa including villus height and villus width were investigated on tissue cross section using a MPIAS-500, a multiple media pathological image analysis system; morphometric indices measurements are expressed as ratio between the data from biopsies on POD 60 and data from day 0, before SBTx.
Statistical Analysis Data are expressed as means ⫾ standard deviation. Means between group were compared over time with two-way analysis of variance test; proportions between study groups were compared applying the chi-square test with the Yeats corrections. Comparisons among the experimental group’s curves were performed using the MANOVA test for repeated measures. Probability values (P) of less than .05 were considered to be statistically significant.
RESULTS
None of the transplanted pigs in the group 3 died before the end of the study; severe infections were the causes of death of two pigs in the group 1 (one pneumonia and one general sepsis occurred on POD 20 and in POD 32, respectively) and one pig in group 2 (pneumonia occurred on POD 18). The overall incidence of acute graft rejection in the tissue specimens examined (biopses along the study and graft samples harvested at the autopsy) was 37.5% for group 1, 23% for group 2, and 13% for group 3 (group 3 vs group 2: P ⬍ 0.05; group 2 and group 3 vs group 1: P ⬍ .001). The nutritional indices of the study groups are shown in the Table 1. Diarrhea was observed in all transplanted piglets, with a mean duration of 15.2 days, 10.4 days, and 7.3 days, respectively, for group 1, group 2, and group 3. The differences between the study groups concerning the duration of the diarrhea, ratio between final and starting weight, and morphometric villi evaluation were statistically significant, showing an advantage for the tranplanted pigs treated in group 3. The HE-stained sections of the epithelial specimen of group 3 intestinal graft harvested in the course of the study confirmed better conservation of the villi and
2026
ZONTA, DONI, ALESSIANI ET AL
Fig 1. D-xylose absorption curve reported study groups compared with control groups.
higher trophic appearance of mucosal structures compared to those of groups 1 and 2. D-xylose absorption curves observed at POD 60 in the treated groups are reported in Fig 1 and are compared with those obtained in the control group. The multivariate analysis of the four data sets applying the MANOVA test showed significant differences along the whole curves of absorption (group 3 vs group 2: P ⫽ .02; group C vs group 3; P ⫽ .04; group 2 vs group 1: P ⫽ .001). DISCUSSION
The preservation-reperfusion injury represents the most critical event during SBTx that may affect the integrity of the mucosal barrier and promote postoperative complications. The consequences of this damage are evident both on the metabolic status of the recipients and on the immunological surveillance. Fluid hypersecretion with loss of electrolytes and proteins is the result of the breakdown of the intestinal epithelium, which is associated to the release of inflammatory mediators acting on the recruitment of the antigen-presenting cells and predisposing to acute cellular rejection. The rupture of the mucosal barrier promotes bacterial translocation, causing severe infectious episodes within a transplanted organism even weakened by hypercatabolic state. The main postoperative complications— namely pneumonia, peritonitis, intestinal leakage, sepsis, ACR, and graft-versus-host disease— have the same root causes, recognizing a unique pathogenetic mechanism that could be improved with preservation of the mucosal barrier. EN was recently approved for enhancing resistance to infection in experimental models and in clinical settings; it
exerts a protective effect to immunity and nutritional status, thus influencing the rate of complication and prognosis. The employment of polymeric EN associated with the immunonutrition such as glutamine and lactobacilli showed a slight advantage toward standard EN solution.1,2 The rationale of early EN is a faster recovery of the mucosal barrier from operative trauma and prevention of starvation leading to bacterial overgrowth, endotoxemia, and release of mediators that could delay the restoration of the barrier function. The complexity of the nutrient that constitutes the EN and the number of enzymatic reactions required for the complete absorption of the substrate may result in a limiting factor reducing the efficacy of the enteral solution. Elemental enteral solution represents a new option for warranting a complete proteinic uptake when the loss of the barrier integrity may reduce the enzymatic surface activities. Our study demonstrated that the availability of elemental nutritional substrates improved the recovery of the absorptive function, highlighting that only the basic functions are preserved after SBTx. The restoration of the absorptive activity may be influenced by the quality of the nutrient. Moreover, the prevalence of easily to metabolize substrates may promptly interrupt the hypercathabolic status.
REFERENCES 1. Schulz RJ, Dignass A, Pascher A, et al: New dietary concepts in small bowel transplantation. Transplant Proc 34:893, 2002
ELEMENTAL ENTERAL NUTRITION 2. Mueller AR, Pasher A, Schulz AR, et al: Clinical small bowel transplantation: focus on mucosal function. Transplant Proc 43: 926, 2002 3. Zhang XQ, Li JS, Li N, et al: Trophic effect of enteral rehabilitative therapy in rat small bowel transplantation. Transplant Proc 37:2351, 2005
2027 4. Alessiani M, Spada M, Merli M, et al: Different models of intestinal transplantation in pigs: technical aspects. Transplant Proc 28:2720, 1996 5. Wu T, Abu Elmagd K, Bonf J, et al: A schema for histologic grading of small intestine allograft acute rejection. Transplantation 75:1241, 2003