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Intestinal Preservation Injury: A Comparison Between Different Rat Strains
Intestinal Preservation Injury: A Comparison Between Different Rat Strains R. Olofsson, M. Oltean, and M. Olausson ABSTRACT Aims. Comparison between the extensive information on intestinal preservation injury in the rat is challenging since various preservation solutions, technical details, and grading systems have been used. This study investigates if strain represents another relevant variable for preservation injury outcome. Methods. Grafts from Piebald-Viral-Glaxo (PVG), Lewis, Brown Norway (BN), Wistar, and Sprague-Dawley (SD) male rats (n ⫽ 8/strain) were used. Grafts were perfused and stored at 4°C in University of Wisconsin (UW) solution (with or without luminal preservation). Intestinal histology was evaluated at 8, 16, and 24 hours of preservation using the Park score. Lactate, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and glucose were measured in the preservation solution. Results. Grafts from PVG and Lewis showed significantly lower injury scores compared to BN, Wistar, and SD at 8 and 16 hours, a difference that disappeared at 24 hours of preservation. Luminal preservation significantly reduced the injury score for all strains, except SD, at 8 and at 16 hours, a difference that disappeared at 24 hours. Biochemical analyses of LDH and lactate levels show a similar pattern both between different strains and the effect of luminal preservation, although not statistically significant. Conclusion. Different rat strains seem to have different susceptibility to intestinal preservation injury under identical conditions. These results indicate that PVG and Lewis are more resistant to preservation injury than Wistar, BN, or SD. The beneficial effect of luminal preservation with UW solution is limited after 8 to 16 hours.
I
NTESTINAL TRANSPLANTATION has emerged as a valid option for patients with intestinal failure presenting complications following long-term total parenteral nutrition. Major improvements have been achieved in recent years within the technical and immunosuppressive field, but still the graft survival remains low, with major mortality due to sepsis and allograft rejection.1 The intestine is extremely susceptible to ischemia, and improved preservation is an essential factor for successful intestinal transplantation. The maximal preservation time is 6 to 8 hours when using University of Wisconsin (UW) solution, although recent experiments suggest that luminal preservation, amino acid addition, and supplementary oxygen delivery could prolong this period.2 Comparison between the extensive information on intestinal preservation injury in the rat is challenging since various preservation solutions, technical details, and grading systems have been used.3–5 The aim of this study
was to determine if strain represents another relevant variable for preservation injury in the rat. MATERIALS AND METHODS Forty male rats from five different strains (Piebald-Viral-Glaxo [PVG], Lewis, Brown Norway [BN], Wistar, and Sprague-Dawley [SD], n ⫽ 8/strain were used. The experiment was approved by the local ethical committee for laboratory animal research. Rats were 10 weeks of age and fasted 24 hours prior to intestinal graft harvesting. Grafts underwent vascular perfusion in situ with cold University of Wisconsin (UW) solution, thereafter harvested and flushed luminally with saline. From each rat six jejunal segments (3 cm each) were harvested and weighed. The segments were divided From the Department of Transplantation and Liver Surgery, Sahlgrenska University Hospital, Göteborg, Sweden. Address reprint requests to Roger Olofsson, MD, Department of Transplantation and Liver Surgery, Sahlgrenska University Hospital, 413 45, Göteborg, Sweden. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.05.046
Transplantation Proceedings, 38, 1789 –1791 (2006)
1789
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OLOFSSON, OLTEAN, AND OLAUSSON
Fig 1. Preservation injury measured according to the Park scale for five different rat strains (PVG, Lewis, BN, Wistar, and SD) with luminal preservation (L) or without luminal preservation (NL) at three different time points (8, 16, and 24 hours). into two groups, luminal preservation (L) or nonluminal preservation (NL); where segments without luminal contact were ligated on both ends to prevent UW solution entry into the lumen. The segments were then preserved at 4°C in UW solution for 8, 16, or 24 hours, respectively. The preserved intestines were fixed in 10% formalin, paraffin embedded and stained with hematoxylin and eosin. The intestinal histology was blindly examined by two observers and scored according to the Park scale.5 Lactate, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and glucose in the preservation solution were measured spectrophotometrically and expressed as units per gram of tissue. All results are expressed as mean ⫾ SD and statistical analyses were performed with analysis of variance and Student t test. Differences were considered significant at P values less than .05.
RESULTS
Histology using the Park scale showed a significant gradual increase in morphological injury with time for all strains regardless of UW luminal presence. Segments from PVG and Lewis showed significantly lower injury score compared to BN, Wistar and SD at 8 hours and 16 hours in the group without luminal preservation. Within the luminal preservation group the injury level was significantly lower when comparing PVG and Lewis with Wistar and SD at both 8
hours and 16 hours. Luminal preservation significantly reduced the injury score for all strains at 8 hours (0.76⫾0.27 U) and at 16 hours except for SD (0.87⫾0.34 U), a difference that disappeared at 24 hours (Fig 1). Results from biochemical analyses of LDH, ALP and lactate show a similar trend compared with morphological results between different strains, although not always statistically significant. Glucose levels increased with preservation time but did not differ between strains. When comparing luminal with non-luminal preservation there was a significant increase in both glucose (except BN and Wistar at 8 hours and Lewis at 16 hours) and ALP (except Lewis at 8 hours) levels and a clear trend towards decreased levels of LDH at 8, 16 and 24 hours (Table 1). DISCUSSION
The extensive yet inconsistent results on intestinal preservation injury in rats are probably due to the use of various preservation solutions, technical details, and grading systems. Our aim was to investigate whether strain represents another variable to consider and this study showed evident morphological differences at 8 hours and 16 hours between
Table 1. Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP), Lactate, and Glucose Levels Measured in UW Solution at Three Different Time Points (8, 16, and 24 Hours) With Luminal (L) or Without Luminal (NL) Preservation UW LDH
UW ALP
UW Lactate
UW Glucose
Strain
8h
16 h
24 h
8h
16 h
24 h
8h
16 h
24 h
PVG (NL) PVG (L) Lewis (NL) Lewis (L) BN (NL) BN (L) Wistar (NL) Wistar (L) SD (NL) SD (L)
103 ⫾ 28 85 ⫾ 9 100 ⫾ 8 83 ⫾ 7 125 ⫾ 3 151 ⫾ 28 165 ⫾ 15 64 ⫾ 5 116 ⫾ 16 111 ⫾ 20
118 ⫾ 21 78 ⫾ 8 132 ⫾ 7 109 ⫾ 41 178 ⫾ 13 134 ⫾ 4 199 ⫾ 45 99 ⫾ 10 162 ⫾ 16 166 ⫾ 32
169 ⫾ 16 86 ⫾ 4 160 ⫾ 7 134 ⫾ 5 182 ⫾ 33 141 ⫾ 20 186 ⫾ 9 108 ⫾ 29 186 ⫾ 19 214 ⫾ 5
0.6 ⫾ 0.1 1.6 ⫾ 0.8 0.8 ⫾ 0.3 1.6 ⫾ 0.8 1.0 ⫾ 0.3 1.9 ⫾ 0.9 1.1 ⫾ 0.6 1.6 ⫾ 0.7 0.8 ⫾ 0.2 2.5 ⫾ 1.3
0.8 ⫾ 0.1 3.4 ⫾ 1.6 1.0 ⫾ 0.2 1.3 ⫾ 0.2 1.0 ⫾ 0.1 2.6 ⫾ 0.2 0.7 ⫾ 0.1 2.8 ⫾ 1.0 1.2 ⫾ 0.4 5.1 ⫾ 0.3
0.8 ⫾ 0.1 4.5 ⫾ 2.1 0.7 ⫾ 0.1 1.8 ⫾ 0.2 1.8 ⫾ 0.2 3.3 ⫾ 1.2 0.8 ⫾ 0.1 3.7 ⫾ 1.1 1.2 ⫾ 0.7 6.3 ⫾ 0.4
9⫾5 8⫾1 15 ⫾ 4 9⫾1 22 ⫾ 3 11 ⫾ 1 7⫾1 6⫾1 9⫾2 10 ⫾ 2
14 ⫾ 1 13 ⫾ 1 26 ⫾ 1 18 ⫾ 2 24 ⫾ 3 22 ⫾ 1 13 ⫾ 2 13 ⫾ 3 18 ⫾ 1 20 ⫾ 1
25 ⫾ 4 24 ⫾ 6 35 ⫾ 8 28 ⫾ 3 34 ⫾ 4 41 ⫾ 5 19 ⫾ 3 21 ⫾ 3 27 ⫾ 4 25 ⫾ 10
8h
16 h
24 h
16 ⫾ 9 24 ⫾ 10 36 ⫾ 5 44 ⫾ 18 85 ⫾ 13 100 ⫾ 6 23 ⫾ 8 33 ⫾ 7 39 ⫾ 10 40 ⫾ 12 42 ⫾ 8 53 ⫾ 13 17 ⫾ 3 24 ⫾ 4 36 ⫾ 4 21 ⫾ 4 39 ⫾ 5 62 ⫾ 12 29 ⫾ 9 26 ⫾ 10 37 ⫾ 11 35 ⫾ 13 91 ⫾ 46 95 ⫾ 7 19 ⫾ 4 27 ⫾ 4 37 ⫾ 17 61 ⫾ 28 110 ⫾ 33 185 ⫾ 94
INTESTINAL PRESERVATION INJURY
strains. Biochemically there were also differences found with a trend that PVG and Lewis had lower LDH, lactate and ALP levels. Though it is hard to formulate an absolute ranking it is clear that PVG and Lewis seem more resistant whereas Wistar, BN and SD seem to be the more sensitive strains. The positive effect of luminal preservation has been shown earlier and these results gives further prove of this finding up to 16 hours of preservation. Concerning LDH and ALP when comparing effects of luminal preservation, contradictory results were found. LDH is a seromuscular enzyme known to rise early in intestinal ischemia6 and luminal preservation in this study seems to be beneficial with decreased levels of LDH. On the other hand ALP showed increased levels for all strains in the luminally preserved group, a finding inconsistent with other both morphological and biochemical results in this study. The probable explanation is that ALP being a mucosal enzyme and hence easier diffuses into the preservation solution when there is a luminal contact. Together with ALP also glucose levels were increased in the luminal preservation group, this probably being due to mucosal glycosidases
1791
cleaving hydroxyl-ethyl starch in the luminal UW solution into glucose. In summary this experiment shows that different rat strains might reveal different susceptibility to intestinal preservation injury under identical preservation conditions, a noteworthy finding when designing and comparing studies in the future. REFERENCES 1. Grant D, Abu-Elmagd K, Reyes J, et al: 2003 report of the intestine transplant registry: a new era has dawned. Ann Surg 241:607, 2005 2. Salehi P, Zhu JZ, Castillo EG, et al: Preserving the mucosal barrier during small bowel storage. Transplantation 76:911, 2003 3. Quaedackers JS, Beuk RJ, Bennet L, et al: An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant Proc 32:1307, 2000 4. Muller AR, Nalesnik M, Platz KP, et al: Evaluation of preservation conditions and various solutions for small bowel preservation. Transplantation 57:649, 1994 5. Park PO, Haglund U, Bulkley GB, et al: The sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion. Surgery 107:574, 1990 6. Thompson JS, Bragg LE, West WW: Serum enzyme levels during intestinal ischemia. Ann Surg 211:369, 1990
I
NTESTINAL TRANSPLANTATION has emerged as a valid option for patients with intestinal failure presenting complications following long-term total parenteral nutrition. Major improvements have been achieved in recent years within the technical and immunosuppressive field, but still the graft survival remains low, with major mortality due to sepsis and allograft rejection.1 The intestine is extremely susceptible to ischemia, and improved preservation is an essential factor for successful intestinal transplantation. The maximal preservation time is 6 to 8 hours when using University of Wisconsin (UW) solution, although recent experiments suggest that luminal preservation, amino acid addition, and supplementary oxygen delivery could prolong this period.2 Comparison between the extensive information on intestinal preservation injury in the rat is challenging since various preservation solutions, technical details, and grading systems have been used.3–5 The aim of this study
was to determine if strain represents another relevant variable for preservation injury in the rat. MATERIALS AND METHODS Forty male rats from five different strains (Piebald-Viral-Glaxo [PVG], Lewis, Brown Norway [BN], Wistar, and Sprague-Dawley [SD], n ⫽ 8/strain were used. The experiment was approved by the local ethical committee for laboratory animal research. Rats were 10 weeks of age and fasted 24 hours prior to intestinal graft harvesting. Grafts underwent vascular perfusion in situ with cold University of Wisconsin (UW) solution, thereafter harvested and flushed luminally with saline. From each rat six jejunal segments (3 cm each) were harvested and weighed. The segments were divided From the Department of Transplantation and Liver Surgery, Sahlgrenska University Hospital, Göteborg, Sweden. Address reprint requests to Roger Olofsson, MD, Department of Transplantation and Liver Surgery, Sahlgrenska University Hospital, 413 45, Göteborg, Sweden. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.05.046
Transplantation Proceedings, 38, 1789 –1791 (2006)
1789
1790
OLOFSSON, OLTEAN, AND OLAUSSON
Fig 1. Preservation injury measured according to the Park scale for five different rat strains (PVG, Lewis, BN, Wistar, and SD) with luminal preservation (L) or without luminal preservation (NL) at three different time points (8, 16, and 24 hours). into two groups, luminal preservation (L) or nonluminal preservation (NL); where segments without luminal contact were ligated on both ends to prevent UW solution entry into the lumen. The segments were then preserved at 4°C in UW solution for 8, 16, or 24 hours, respectively. The preserved intestines were fixed in 10% formalin, paraffin embedded and stained with hematoxylin and eosin. The intestinal histology was blindly examined by two observers and scored according to the Park scale.5 Lactate, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and glucose in the preservation solution were measured spectrophotometrically and expressed as units per gram of tissue. All results are expressed as mean ⫾ SD and statistical analyses were performed with analysis of variance and Student t test. Differences were considered significant at P values less than .05.
RESULTS
Histology using the Park scale showed a significant gradual increase in morphological injury with time for all strains regardless of UW luminal presence. Segments from PVG and Lewis showed significantly lower injury score compared to BN, Wistar and SD at 8 hours and 16 hours in the group without luminal preservation. Within the luminal preservation group the injury level was significantly lower when comparing PVG and Lewis with Wistar and SD at both 8
hours and 16 hours. Luminal preservation significantly reduced the injury score for all strains at 8 hours (0.76⫾0.27 U) and at 16 hours except for SD (0.87⫾0.34 U), a difference that disappeared at 24 hours (Fig 1). Results from biochemical analyses of LDH, ALP and lactate show a similar trend compared with morphological results between different strains, although not always statistically significant. Glucose levels increased with preservation time but did not differ between strains. When comparing luminal with non-luminal preservation there was a significant increase in both glucose (except BN and Wistar at 8 hours and Lewis at 16 hours) and ALP (except Lewis at 8 hours) levels and a clear trend towards decreased levels of LDH at 8, 16 and 24 hours (Table 1). DISCUSSION
The extensive yet inconsistent results on intestinal preservation injury in rats are probably due to the use of various preservation solutions, technical details, and grading systems. Our aim was to investigate whether strain represents another variable to consider and this study showed evident morphological differences at 8 hours and 16 hours between
Table 1. Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP), Lactate, and Glucose Levels Measured in UW Solution at Three Different Time Points (8, 16, and 24 Hours) With Luminal (L) or Without Luminal (NL) Preservation UW LDH
UW ALP
UW Lactate
UW Glucose
Strain
8h
16 h
24 h
8h
16 h
24 h
8h
16 h
24 h
PVG (NL) PVG (L) Lewis (NL) Lewis (L) BN (NL) BN (L) Wistar (NL) Wistar (L) SD (NL) SD (L)
103 ⫾ 28 85 ⫾ 9 100 ⫾ 8 83 ⫾ 7 125 ⫾ 3 151 ⫾ 28 165 ⫾ 15 64 ⫾ 5 116 ⫾ 16 111 ⫾ 20
118 ⫾ 21 78 ⫾ 8 132 ⫾ 7 109 ⫾ 41 178 ⫾ 13 134 ⫾ 4 199 ⫾ 45 99 ⫾ 10 162 ⫾ 16 166 ⫾ 32
169 ⫾ 16 86 ⫾ 4 160 ⫾ 7 134 ⫾ 5 182 ⫾ 33 141 ⫾ 20 186 ⫾ 9 108 ⫾ 29 186 ⫾ 19 214 ⫾ 5
0.6 ⫾ 0.1 1.6 ⫾ 0.8 0.8 ⫾ 0.3 1.6 ⫾ 0.8 1.0 ⫾ 0.3 1.9 ⫾ 0.9 1.1 ⫾ 0.6 1.6 ⫾ 0.7 0.8 ⫾ 0.2 2.5 ⫾ 1.3
0.8 ⫾ 0.1 3.4 ⫾ 1.6 1.0 ⫾ 0.2 1.3 ⫾ 0.2 1.0 ⫾ 0.1 2.6 ⫾ 0.2 0.7 ⫾ 0.1 2.8 ⫾ 1.0 1.2 ⫾ 0.4 5.1 ⫾ 0.3
0.8 ⫾ 0.1 4.5 ⫾ 2.1 0.7 ⫾ 0.1 1.8 ⫾ 0.2 1.8 ⫾ 0.2 3.3 ⫾ 1.2 0.8 ⫾ 0.1 3.7 ⫾ 1.1 1.2 ⫾ 0.7 6.3 ⫾ 0.4
9⫾5 8⫾1 15 ⫾ 4 9⫾1 22 ⫾ 3 11 ⫾ 1 7⫾1 6⫾1 9⫾2 10 ⫾ 2
14 ⫾ 1 13 ⫾ 1 26 ⫾ 1 18 ⫾ 2 24 ⫾ 3 22 ⫾ 1 13 ⫾ 2 13 ⫾ 3 18 ⫾ 1 20 ⫾ 1
25 ⫾ 4 24 ⫾ 6 35 ⫾ 8 28 ⫾ 3 34 ⫾ 4 41 ⫾ 5 19 ⫾ 3 21 ⫾ 3 27 ⫾ 4 25 ⫾ 10
8h
16 h
24 h
16 ⫾ 9 24 ⫾ 10 36 ⫾ 5 44 ⫾ 18 85 ⫾ 13 100 ⫾ 6 23 ⫾ 8 33 ⫾ 7 39 ⫾ 10 40 ⫾ 12 42 ⫾ 8 53 ⫾ 13 17 ⫾ 3 24 ⫾ 4 36 ⫾ 4 21 ⫾ 4 39 ⫾ 5 62 ⫾ 12 29 ⫾ 9 26 ⫾ 10 37 ⫾ 11 35 ⫾ 13 91 ⫾ 46 95 ⫾ 7 19 ⫾ 4 27 ⫾ 4 37 ⫾ 17 61 ⫾ 28 110 ⫾ 33 185 ⫾ 94
INTESTINAL PRESERVATION INJURY
strains. Biochemically there were also differences found with a trend that PVG and Lewis had lower LDH, lactate and ALP levels. Though it is hard to formulate an absolute ranking it is clear that PVG and Lewis seem more resistant whereas Wistar, BN and SD seem to be the more sensitive strains. The positive effect of luminal preservation has been shown earlier and these results gives further prove of this finding up to 16 hours of preservation. Concerning LDH and ALP when comparing effects of luminal preservation, contradictory results were found. LDH is a seromuscular enzyme known to rise early in intestinal ischemia6 and luminal preservation in this study seems to be beneficial with decreased levels of LDH. On the other hand ALP showed increased levels for all strains in the luminally preserved group, a finding inconsistent with other both morphological and biochemical results in this study. The probable explanation is that ALP being a mucosal enzyme and hence easier diffuses into the preservation solution when there is a luminal contact. Together with ALP also glucose levels were increased in the luminal preservation group, this probably being due to mucosal glycosidases
1791
cleaving hydroxyl-ethyl starch in the luminal UW solution into glucose. In summary this experiment shows that different rat strains might reveal different susceptibility to intestinal preservation injury under identical preservation conditions, a noteworthy finding when designing and comparing studies in the future. REFERENCES 1. Grant D, Abu-Elmagd K, Reyes J, et al: 2003 report of the intestine transplant registry: a new era has dawned. Ann Surg 241:607, 2005 2. Salehi P, Zhu JZ, Castillo EG, et al: Preserving the mucosal barrier during small bowel storage. Transplantation 76:911, 2003 3. Quaedackers JS, Beuk RJ, Bennet L, et al: An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant Proc 32:1307, 2000 4. Muller AR, Nalesnik M, Platz KP, et al: Evaluation of preservation conditions and various solutions for small bowel preservation. Transplantation 57:649, 1994 5. Park PO, Haglund U, Bulkley GB, et al: The sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion. Surgery 107:574, 1990 6. Thompson JS, Bragg LE, West WW: Serum enzyme levels during intestinal ischemia. Ann Surg 211:369, 1990