Functional and Histological Comparison of Rat Liver Preserved in University of Wisconsin Solution Compared with Tissue Preserved in a Novel Solution

Functional and Histological Comparison of Rat Liver Preserved in University of Wisconsin Solution Compared with Tissue Preserved in a Novel Solution C.L. Corps, I. Ahmed, S. McKenzie, M. Shires, D.J. Potts, and J.P.A. Lodge ABSTRACT An isolated perfused rat liver model was used to investigate biochemical and histologic changes during 2 hours of reperfusion after 24 hours of cold storage to compare Leeds solution (LS) with University of Wisconsin solution (UW). Compared with livers stored in UW, those perfused with LS showed significantly higher bile flow and lower enzyme production (P ⬍ .05 by 1-way analysis of variance). For example, after 120 minutes, alanine aminotransferase results were: LS 38.9 U/L vs UW 66.8 U/L and bile flows were LS 10.3 ␮g/15 min/g liver vs UW 9.2 ␮g/15 min/g liver. Histologically the reticulin breakdown was greater and its reformation slower in UW-preserved livers. Liver tissue was viable in both groups, as shown by the increased glycogen content after reperfusion in both groups, but seen at a higher rate among LS, perfused livers. In conclusion, LS compared favorably with UW to prevent ischemic damage and so could offer an alternative perfusion medium to UW. everal papers have recently suggested that although histidine-tryptophan-ketoglutarate (HTK) is as good as or better than University of Wisconsin solution (UW) for short periods of cold ischemia, such as in live donation, it was not as efficient for longer periods of cold ischemia, mainly owing to delayed graft function.1–3 Therefore, other alternatives to UW are required. The present study examined biochemical and histologic analyses of rat liver tissue during preservation and after reperfusion, seeking to compare the effectiveness of Leeds solution (LS) with UW, which is widely used in organ preservation. The content of each solution as shown in Table 1

S

METHODS The experiment was carried out under a Home Office Licence according to standard guidelines for animal care. Male Wistar rats (280 –340 g) were anesthetized using a single intraperitoneal injection of pentobarbitone (0.6 ␮g/g). After midline and subcostal laparotomy, the liver was exposed and catheters placed into portal vein and abdominal aorta. The aorta was cut and the liver flushed with either ice cold LS (n ⫽ 24) or UW (n ⫽ 18) before storage for up to 24 hours in 60 mL of the chosen solution at 4°C. After storage, the livers were reperfused for 2 hours using rat blood in an isolated perfusion system, as described by Ahmed et al.4 During this period, collected bile was measured every 15 minutes, to be expressed per gram of liver tissue. Liver enzymes, alanine aminotransferase (ALT), asparatate transaminase (AST), and lactate dehydrogenase (LDH) were determined in hepatic vein samples obtained every 30 minutes during

reperfusion. The samples were tested in a Unicam 8625UV/VIS spectrophotometer. Histologic examinations of liver samples obtained before and after reperfusion were placed in glutaraldehyde. Paraformaldehyde-fixed tissue embedded in paraffin was cut at 4 ␮m and stained using Periodic acid–Schiff (PAS) or Gordon-Sweet stain. The PAS reaction was used as a useful indicator of the presence of tissue carbohydrates, particularly glycogen, which stained magenta. The reaction intensity and color depended on the length of treatment with PAS.5 Glycogen, the store of readily available glucose in the liver, is considered to be the main buffer of blood glucose levels. Glycogen can disappear from hepatocytes for 2 reasons: first to replenish the level of glucose in the blood; or second, owing to hepatocyte injury, which causes its loss at an early stage. The Gordon-Sweet stain visualizes tissue reticulin fibers. These fine delicate fibers (0.5–2.0 ␮m in diameter) provide the bulk of the supporting framework in cellular organs such as the liver. They are arranged in a 3-dimensional structure to provide a system of individual cell support; historically, they were considered to be immature collagen fibres. Histologic differentiation is by silver impregnation techniques, which stain collagen brown/mauve and reticulin black.5

From the Transplant Science Group, Clinical Sciences Building, St. James’s University Hospital, Leeds, United Kingdom. Address reprint requests to Dr C. L. Corps, Transplant Science Group, Department of Hepatology and Transplantation, St. James’s University Hospital, Leeds, West Yorkshire, LS9 7TF, UK. E-mail: [email protected]

© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/–see front matter doi:10.1016/j.transproceed.2010.06.029

Transplantation Proceedings, 42, 3427–3430 (2010)

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CORPS, AHMED, MCKENZIE ET AL Table 1. Preservation Solution Components Component

LS

Lactobionate (mmol/L) Sucrose (mmol/L) Raffinose (mmol/L) PEG (mmol/L) HES (g/L) Na2HPO4 (mmol/L) NaH2PO4 · 2H2O (mmol/L) K2HPO4 (mmol/L) MgSO4 (mmol/L) Glutathione (mmol/L) Glutamine (mmol/L) Allopurinol (mmol/L) Adenosine (mmol/L) Acetylsalicylic acid (mmol/L) Nicardipine (mmol/L) Penicillin (U) Insulin (U) Dexamethosone (mg) Osmolarity (mOsmol/L) pH

UW

50 100

100 30

1 50 26.45 16.66 25 25 3

3 20 0.4

1 5

Fig 2. Aspartate transaminase (AST) released from the livers after preservation with either LS (solid bars) or UW (open bars). LS-preserved livers released a statistically lower level of AST (P ⬍ .05; 1-way ANOVA) than UW-perfused livers throughout the experiment.

0.5 0.005 40 100 8 320 7.4

350 7.0

Fig 3. Alanine aminotransferase (ALT) released from the livers after preservation with either LS (solid bars) or UW (open bars). LS-preserved livers released a statistically lower level of ALT (P ⬍ .05; 1-way ANOVA) than UW-perfused livers throughout the experiment.

Fig 1. Bile flow released from the livers after preservation with either LS (solid squares) or UW (open squares). LS-preserved livers maintained a statistically higher bile flow rate (P ⬍ .05; 1-way ANOVA) than UW-perfused livers up to the 105–120minute time point. Table 2. Bile Flow (␮g/15 min/g Liver) Time (min)

LS SEM UW SEM

0–15

15–30

30–45

45–60

60–75

75–90

90–105

105–120

5.6 0.4 4.6 0.9

10.8 0.4 8.1 0.8

12.6 0.4 8.9 1.1

14.9 0.5 9.1 1.2

14.1 0.4 11.1 0.7

13.1 0.5 10.3 1

12.2 0.4 9.8 1.1

10.3 0.3 9.2 1.3

Fig 4. Lactate dehydrogenase (LDH) released from the livers after preservation with either LS (solid bars) or UW (open bars). LS-preserved livers released a lower level of ALT than UWperfused livers throughout the experiment, which reached a significant difference in the rinse (P ⬍ 0.05; 1-way ANOVA). Table 3. Liver Enzymes (U/L)

RESULTS

After reperfusion, bile flow (Fig 1 and Table 2) was significantly greater in livers perfused with LS than with UW (P ⬍ .05; 1-way analysis of variance [ANOVA]). All liver enzymes (AST, ALT, and LDH; Figs 2– 4 and Table 3) were released from livers stored in LS at significantly lower levels than from livers stored in UW (P ⬍ .05; 1-way ANOVA). After 24 hours of cold storage, PAS stain showed a slower rate of disappearance of glycogen from the hepa-

Time (min)

LS AST ALT LDH UW AST ALT LDH

Rinse

30

60

90

120

39.8 42.9 411 68.2 62.1 612

14.2 17.7 104 22.4 28.1 232

19.9 26.7 199 50.3 37.9 281

32.7 32.1 235 78.7 50.8 310

45.4 38.9 325 89.2 66.8 393

RAT LIVER IN NOVEL VERSUS UW SOLUTION

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Fig 5. (A) LS-perfused tissue after 24 hours of cold storage: glycogen present in some cells. (B) UW-perfused tissue after 24 hours of cold storage: no glycogen present in any cells. (C) LS-perfused tissue after 2 hours of reperfusion: glycogen present in many cells. (D) UW-perfused tissue after 2 hours of reperfusion: very little glycogen in cells.

tocytes of livers preserved in LS than in UW (Fig 5A and B). Glycogen levels recovered to a greater degree in hepatocytes of livers which had been perfused with LS than with UW after 2 hours of reperfusion with rat red cells (Fig 5C and D). Reticulin fraying and condensation were widespread after 24 hours of liver cold storage in either LS or UW. In UW, liver cellular differentiation was lost, although it was still present in LS specimens (Fig 6A and B). After 2 hours of reperfusion, reticulin was seen around the portal triad and central venules in livers stored in either solution. Livers perfused with LS showed reticulin also between cells, therefore showing sinusoids, a finding that was not present in UW-perfused livers (Fig 6C and D). DISCUSSION

After reperfusion, there was significantly lower release of all liver enzymes and higher bile flow rate organs perfused in LS versus UW, indicating better preservation by LS. PAS histology examined hepatic glycogen content, which when degraded becomes glucose, the substrate that allows responsiveness of the tricarboxylic acid cycle upon oxygenation.

The glycogen in reperfused LS-treated livers can come from 2 sources: first, residual glycogen remaining after 24 hours of storage (Fig 5A); and second, de novo generated after reperfusion; indicating viable hepatocytes. Because there was no glycogen present after 24 hours of storage in UW (Fig 5B) the small amount of glycogen must have been produced by the hepatocytes after reperfusion, demonstrating that these hepatocytes were also viable. Earlier work by our laboratory6 showed that during preservation the glycogen content in UW-perfused livers remained at a higher level than in HTK-perfused liver cells. The present work showed that although the level of glycogen in UW-preserved livers started to recover by 2 hours after reperfusion, the level in LS-perfused livers was depleted less severely and recovered at a quicker rate. This suggests that the delayed graft function that sometimes occurs when HTK is used to perfuse livers should not be seen in organs perfused with LS, in the same way that it is rarely seen with UW-perfused livers. Reticulin is made up of fine delicate 0.5–2.0-␮m-diameter fibers, that provide the bulk of the supporting framework or scaffold for hepatic tissue. It breaks down with irregular

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Fig 6. (A) LS-perfused tissue after 24 hours of cold storage: widespread fraying and condensing of reticulin fibres, but cellular differentiation still visible. (B) UW-perfused tissue after 24 hours of cold storage: widespread fraying and condensing of reticulin fibres; loss cellular differentiation. (C) LS-perfused tissue after 2 hours of reperfusion: reticulin seen around the portal triad and central venue, also between cells, showing sinusoids. (D) UW-perfused tissue after 2 hours of reperfusion: Reticulin seen around portal triad and central venule, but little is seen in the sinusoids.

degenerative changes, causing loss of connections with the axial reticulin and giving appearances like a “fraying rope.”7 The more the reticulin is frayed, the longer it will take to re-form. The present observations suggest that reticulin did not break down any further after reperfusion and started to re-form fairly quickly. Because reticulin was broken to a greater extent in livers perfused with UW compared with LS (Fig 6A and B), it was hardly surprising that it took longer for it to be re-formed in livers perfused with UW (Fig 6D) than with LS (Fig 6C), which is consistent with the biochemical analysis. These observations support the functional studies of Ahmed et al.8 and Corps et al.9 which support the view that LS compares favorably with UW to prevent ischemic damage and so could be an alternative perfusion solution to either UW or HTK. REFERENCES 1. Lynch RJ, Kubus J, Chenault RH, et al: Comparison of histidine-tryptophan-ketoglutarate and University of Wisconsin preservation in renal transplantation. Am J Transplant 8:567, 2008

2. van Gulik TM, Reinders ME, Nio R, et al: Preservation of canine liver grafts using histidine-tryptophan-ketoglutarate solution. Transplantation 57:167, 1994 3. Jain A, Mohanka R, Orloff M, et al: University of Wisconsin versus histidine-tryptophan-ketoglutarate for tissue preservation in live-donor liver transplantation. Exp Clin Transplant 4:451, 2006 4. Ahmed I, Attia M, Ahmad N, et al: Use of isolated perfused rat liver model for testing liver preservation solutions. Transplant Proc 33:3709, 2001 5. Bancroft J, Gamble M: Theory and practice of histological techniques. New York: Churchill Livingstone; 2002 6. Corps CL, Shires M, Crellin D, et al: HTK: why does it appear to cause delayed graft function following prolonged cold ischaemia? Transplant Proc 41:3567, 2009 7. Gross P, McNeney JM, Babyak MA: Experimental silicosis: a model for the study of inflammatory pulmonary reticulin. Chest 43:113, 1963 8. Corps CL, Shires M, Crellin D, et al: The influence on energy kinetics and histology of different preservation solutions seen during cold ischaemia in the liver. Transplant Proc 41:4088, 2009