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In vivo changes in acute rejection of rat small bowel allografts
In Vivo Changes in Acute Rejection of Rat Small Bowel Allografts T. Stojanovic, R. Schlemminger, J. Bedke, H.-J. Gro¨ne, M. Heuser, I. Leister, M. Hecker, H. Becker, and P.M. Markus
S
MALL bowel transplantation (SBTX) has become a clinical reality. Although 1-year patient and graft survival has increased, still 40% of grafts fail within this early period. Among the severest problems in SBTX is acute graft rejection.1 Histology is the golden standard in the diagnosis of acute rejection in SBTX showing first histological signs of rejection on postoperative day (POD) 6.2,3 Recently some Investigators have shown that noninvasive semiquantitative video imaging of graft mucosa is a helpful method to detect graft rejection.4,5 Because little is known about the in vivo effects of acute rejection on microcapillary perfusion, we analyzed the influence of acute rejection on microcapillary perfusion in rat small bowel grafts by means of in vivo microscopy.
Microcirculatory Evaluation Quantitative analysis of microhemodynamics included the determination of the perfusion index (PI) (% of all villi perfused ⫹ 0.5⫻% of all irregularly perfused villi; magnification; 243⫻), the stasis index (SI) (% of all nonperfused villi; magnification; 243⫻), and the functional capillary density (FCD) (length of perfused nutritive capillaries per villus area [1/cm]; magnification: 476⫻.) The FCD was assessed by computer-assisted image analysis CAPIMAGEProgram (Zeintl, Heidelberg, Germany).
Statistics The Mann-Whitney U test was used for the calculation of statistical significance. P ⬍ .01 was considered statistical significant. Values expressed as mean and standard error of the mean.
RESULTS Perfusion Index MATERIALS AND METHODS Animals Heterotopic SBTX was performed in the fully allogeneic BN (RT1n) to LEW (RT1l) and syngeneic LEW to LEW rat strain combination. In vivo microscopy was performed from POD 1 to POD 7 in allogeneic and syngeneic groups. Physiologic nontransplanted animals served as additional control. The experimental groups consisted of six animals each group for POD 1, 6, and 7, and three animals each group for POD 2, 3, and 4.
In Vivo Microscopy For in vivo microscopy, a jejunal section of the transplanted bowel was exteriorised on a specially designed mechanical stage attached to the microscope. The stage was heated to avoid hypothermia and the tissue was superfused with Ringer’s lactate to avoid drying. To study the mucosal microcirculation a longitudinal incision was placed at the antimesenteric border of the bowel wall. In vivo microscopy was performed using a Zeiss Axiotech Vario 100 for epi-illumination with a 100-HBO mercury lamp with 10⫻ (long distance), 20⫻ and 40⫻ (water immersion) objectives; a magnification of 243⫻, 476⫻, and 933⫻ was reached. The observations were recorded by means of a charge coupled device video camera (CF 8/1, Kappa, Gleichen, Germany) and transferred to a video system for off-line evaluation. The intestinal microcirculation was visualized after injection of 0.8 mL 0.5% flourescein isothyocyanate-labeled dextran (Sigma, St Louis, Mo). In each transplant a minimum of 10 regions of interest were analyzed to obtain representative values for each parameter.
In the syngeneic group the PI did not change significantly compared to physiologic control animals. In the allogeneic group the PI started to decline significantly from POD 4 compared to syngeneic group (al. 0.78 ⫾ 0.033 vs. sy. 0.96 ⫾ 0.0065; P ⬍ .01) with a total arrest of perfusion on POD 7 (al. 0.01 ⫾ 0.004 vs. sy. 0.96 ⫾ 0.0095; P ⬍ .01). Stasis Index
Due to good perfusion the SI in the syngeneic and physiologic group had an index level of 0 over the total study course of 7 days. In the allogeneic group the SI started to increase from POD 5 (0.12 ⫾ 0.03; P ⬍ .01), reaching a “breakpoint of perfusion” on POD 6 with 50% stasis of villus perfusion (0.48 ⫾ 0.06; P ⬍ .01) to almost 100% nonperfused villi on POD 7 (0.97 ⫾ 0.008; P ⬍ .01). Function Capillary Density
The FCD in the syngeneic group did not change significantly over POD, 1 to 7 compared to physiologic controls. From the Departments of Surgery (T.S., R.S., J.B., M.H., I.L., H.B., P.M.M.), and Cardiovascular Physiology (M.H.), University of Go¨ttingen, Go¨ttingen, and German Cancer Research Centre/ Department of Cellular and Molecular Pathology (H.-J.G.), University of Heidelberg, Heidelberg, Germany. Address reprint requests to Dr T. Stojanovic, University of Gottingen, Abt1. Allgemeinchirurgie, Robert Koch Str. 40, 37073 Gottingen, Sweden.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01208-2
Transplantation Proceedings, 32, 1247–1248 (2000)
1247
1248
In the allogeneic group the FCD decreased significantly from POD 4 (al. 594 ⫾ 9 vs. sy. 804 ⫾ 14) to a minimum on day 7 (al. 32 ⫾ 8 vs. sy. 812 ⫾ 14 [cm⫺1]; P ⬍ .01). SUMMARY
Microcirculation was not altered in syngeneic SBTX and resembled that of physiologic controls over the whole examination period. In contrast allogeneic SBTX showed severe alterations in FCD, PI, and SI beginning on POD 4 to a maximum of alterations on POD 7 with total breakdown of villus perfusion. CONCLUSION
By means of in vivo microscopy functional changes can be seen before major histologic changes appear. In the experimental setting changes are observed between POD 4 and
STOJANOVIC, SCHLEMMINGER, BEDKE ET AL
POD 6. After POD 6 graft perfusion has ceased and changes are more likely attributed to beginning graft necrosis than rejection. In vivo microscopy could serve as a new sensitive marker for transplant rejection in addition to the gold standard of histology. The clinical significance of our findings await further investigation and mainly depend on the development of nontoxic fluorescent dyes. REFERENCES 1. Grant D: Transplantation 67:1061, 1999 2. Rosemurgy AS, Schraut WH: Am J Surg 151:XX, 1986 3. Schmid T, Oberhuber G, Ko ¨ro ¨zsi G, et al: Gastoenterology 96:1529, 1989 4. Timmermann W, Hoppe H, Otto C, et al: Transplantation 67:1555, 1999 5. Kato T, O’Brien CB, Nishida S, et al: Gastrointest Endosc 50:257, 1999
S
MALL bowel transplantation (SBTX) has become a clinical reality. Although 1-year patient and graft survival has increased, still 40% of grafts fail within this early period. Among the severest problems in SBTX is acute graft rejection.1 Histology is the golden standard in the diagnosis of acute rejection in SBTX showing first histological signs of rejection on postoperative day (POD) 6.2,3 Recently some Investigators have shown that noninvasive semiquantitative video imaging of graft mucosa is a helpful method to detect graft rejection.4,5 Because little is known about the in vivo effects of acute rejection on microcapillary perfusion, we analyzed the influence of acute rejection on microcapillary perfusion in rat small bowel grafts by means of in vivo microscopy.
Microcirculatory Evaluation Quantitative analysis of microhemodynamics included the determination of the perfusion index (PI) (% of all villi perfused ⫹ 0.5⫻% of all irregularly perfused villi; magnification; 243⫻), the stasis index (SI) (% of all nonperfused villi; magnification; 243⫻), and the functional capillary density (FCD) (length of perfused nutritive capillaries per villus area [1/cm]; magnification: 476⫻.) The FCD was assessed by computer-assisted image analysis CAPIMAGEProgram (Zeintl, Heidelberg, Germany).
Statistics The Mann-Whitney U test was used for the calculation of statistical significance. P ⬍ .01 was considered statistical significant. Values expressed as mean and standard error of the mean.
RESULTS Perfusion Index MATERIALS AND METHODS Animals Heterotopic SBTX was performed in the fully allogeneic BN (RT1n) to LEW (RT1l) and syngeneic LEW to LEW rat strain combination. In vivo microscopy was performed from POD 1 to POD 7 in allogeneic and syngeneic groups. Physiologic nontransplanted animals served as additional control. The experimental groups consisted of six animals each group for POD 1, 6, and 7, and three animals each group for POD 2, 3, and 4.
In Vivo Microscopy For in vivo microscopy, a jejunal section of the transplanted bowel was exteriorised on a specially designed mechanical stage attached to the microscope. The stage was heated to avoid hypothermia and the tissue was superfused with Ringer’s lactate to avoid drying. To study the mucosal microcirculation a longitudinal incision was placed at the antimesenteric border of the bowel wall. In vivo microscopy was performed using a Zeiss Axiotech Vario 100 for epi-illumination with a 100-HBO mercury lamp with 10⫻ (long distance), 20⫻ and 40⫻ (water immersion) objectives; a magnification of 243⫻, 476⫻, and 933⫻ was reached. The observations were recorded by means of a charge coupled device video camera (CF 8/1, Kappa, Gleichen, Germany) and transferred to a video system for off-line evaluation. The intestinal microcirculation was visualized after injection of 0.8 mL 0.5% flourescein isothyocyanate-labeled dextran (Sigma, St Louis, Mo). In each transplant a minimum of 10 regions of interest were analyzed to obtain representative values for each parameter.
In the syngeneic group the PI did not change significantly compared to physiologic control animals. In the allogeneic group the PI started to decline significantly from POD 4 compared to syngeneic group (al. 0.78 ⫾ 0.033 vs. sy. 0.96 ⫾ 0.0065; P ⬍ .01) with a total arrest of perfusion on POD 7 (al. 0.01 ⫾ 0.004 vs. sy. 0.96 ⫾ 0.0095; P ⬍ .01). Stasis Index
Due to good perfusion the SI in the syngeneic and physiologic group had an index level of 0 over the total study course of 7 days. In the allogeneic group the SI started to increase from POD 5 (0.12 ⫾ 0.03; P ⬍ .01), reaching a “breakpoint of perfusion” on POD 6 with 50% stasis of villus perfusion (0.48 ⫾ 0.06; P ⬍ .01) to almost 100% nonperfused villi on POD 7 (0.97 ⫾ 0.008; P ⬍ .01). Function Capillary Density
The FCD in the syngeneic group did not change significantly over POD, 1 to 7 compared to physiologic controls. From the Departments of Surgery (T.S., R.S., J.B., M.H., I.L., H.B., P.M.M.), and Cardiovascular Physiology (M.H.), University of Go¨ttingen, Go¨ttingen, and German Cancer Research Centre/ Department of Cellular and Molecular Pathology (H.-J.G.), University of Heidelberg, Heidelberg, Germany. Address reprint requests to Dr T. Stojanovic, University of Gottingen, Abt1. Allgemeinchirurgie, Robert Koch Str. 40, 37073 Gottingen, Sweden.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01208-2
Transplantation Proceedings, 32, 1247–1248 (2000)
1247
1248
In the allogeneic group the FCD decreased significantly from POD 4 (al. 594 ⫾ 9 vs. sy. 804 ⫾ 14) to a minimum on day 7 (al. 32 ⫾ 8 vs. sy. 812 ⫾ 14 [cm⫺1]; P ⬍ .01). SUMMARY
Microcirculation was not altered in syngeneic SBTX and resembled that of physiologic controls over the whole examination period. In contrast allogeneic SBTX showed severe alterations in FCD, PI, and SI beginning on POD 4 to a maximum of alterations on POD 7 with total breakdown of villus perfusion. CONCLUSION
By means of in vivo microscopy functional changes can be seen before major histologic changes appear. In the experimental setting changes are observed between POD 4 and
STOJANOVIC, SCHLEMMINGER, BEDKE ET AL
POD 6. After POD 6 graft perfusion has ceased and changes are more likely attributed to beginning graft necrosis than rejection. In vivo microscopy could serve as a new sensitive marker for transplant rejection in addition to the gold standard of histology. The clinical significance of our findings await further investigation and mainly depend on the development of nontoxic fluorescent dyes. REFERENCES 1. Grant D: Transplantation 67:1061, 1999 2. Rosemurgy AS, Schraut WH: Am J Surg 151:XX, 1986 3. Schmid T, Oberhuber G, Ko ¨ro ¨zsi G, et al: Gastoenterology 96:1529, 1989 4. Timmermann W, Hoppe H, Otto C, et al: Transplantation 67:1555, 1999 5. Kato T, O’Brien CB, Nishida S, et al: Gastrointest Endosc 50:257, 1999