Modulation of microcirculatory changes in the late phase of hepatic ischaemia-reperfusion injury by remote ischaemic preconditioning

Abstract

Background: Remote ischaemic preconditioning (RIPC) is a novel method of protecting the liver from ischaemia-reperfusion (I-R) injury. Protective effects in the early phase (4-6 h) have been demonstrated, but no studies have focused on the late phase (24 h) of hepatic I-R. This study analysed events in the late phase of I-R following RIPC and focused on the microcirculation, inflammatory cascade and the role of cytokine-induced neutrophil chemoattractant-1 (CINC-1).

Methods: A standard animal model was used. Remote preconditioning prior to I-R was induced by intermittent limb ischaemia. Ischaemia was induced in the left and median lobes of the liver (70%). The animals were recovered after 45 min of liver ischaemia. At 24 h, the animals were re-evaluated under anaesthesia. Hepatic microcirculation, sinusoidal leukocyte adherence and hepatocellular death were assessed by intravital microscopy, hepatocellular injury by standard biochemistry and serum CINC-1 by enzyme-linked immunosorbent assay (ELISA).

Results: At 24 h post I-R, RIPC was found to have improved sinusoidal flow by increasing the sinusoidal diameter. There was no effect of preconditioning on the velocity of red blood cells, by contrast with the early phase of hepatic I-R. Remote ischaemic preconditioning significantly reduced hepatocellular injury, neutrophil-induced endothelial injury and serum CINC-1 levels.

Conclusions: Remote ischaemic preconditioning is amenable to translation into clinical practice and may improve outcomes in liver resection surgery and transplantation.

Figure 1

Design of recovery experiments. RIPC, remote ischaemic preconditioning; isch, ischaemia; rep, reperfusion

Figure 2

Velocity of red blood cell flow in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h. Values are expressed as the mean ± standard deviation. P-value = not significant

Figure 3

Sinusoidal flow in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h, calculated as V × (D/2)2 ×π, where V is the velocity of red blood cells and D is sinusoidal diameter. Better flow was found in preconditioned animals. Values are expressed as the mean ± standard deviation. P-value = not significant

Figure 4

Sinusoidal diameter in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h. A significantly increased diameter was found in the preconditioned group. Values are expressed as the mean ± standard deviation. *P = 0.001 (I–R/RIPC + I–R)

Figure 5

Sinusoidal perfusion in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h. The perfusion index in preconditioned animals was significantly higher than in non-preconditioned animals. Values are expressed as the mean ± standard deviation. *P = 0.007 (I–R/RIPC + I–R)

Figure 6

(a) Venular neutrophil adhesion in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h. Venular neutrophil adhesion was significantly reduced in preconditioned animals compared with non-preconditioned animals. Values are expressed as the mean ± standard deviation. *P = 0.00 (I–R/RIPC + I–R). (b) Sinusoidal neutrophil adhesion in I–R and RIPC + I–R at 24 h. Sinusoidal neutrophil adhesion was significantly reduced in the preconditioned group compared with the non-preconditioned group. Values are expressed as the mean ± standard deviation. *P = 0.00 (I–R/RIPC + I–R)

Figure 7

(a) Hepatocellular cell death in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h by propidium iodide staining. The number of cells divided by the surface area of the field gives the number of cells/mm². Dead cells are stained pink. Hepatocellular cell death in the I–R group (b) was significantly higher than in the RIPC + I–R group (c). Values are expressed as the mean ± standard deviation. *P = 0.00 (I–R/RIPC + I–R). (Original magnification × 40)

Figure 8

Transaminase levels in ischaemia–reperfusion (I–R) and in remote ischaemic preconditioning (RIPC) + I–R at 24 h. Transaminase levels were significantly reduced in preconditioned animals. Values are expressed as mean ± standard deviation. (a) Alanine aminotransferase (ALT) (P = 0.881). (b) Aspartate aminotransferase (AST) (P = 0.630)

Figure 9

Histology in (a) ischaemia–reperfusion (I–R) and (b) remote ischaemic preconditioning (RIPC) + I–R at 24 h. Very severe injury with abundant ballooning degeneration, necrosis and very diffuse and significant neutrophil adhesion is seen in the I–R group. Apoptosis is evident. The RIPC + I–R group shows less injury, with some ballooning and degeneration, as well as neutrophilic infiltration. (Haematoxylin and eosin stain; original magnification × 20)

Figure 10

Serum CINC levels in ischaemia–reperfusion (I–R) and remote ischaemic preconditioning (RIPC) + I–R at 24 h were significantly higher in the I–R group than in the RIPC + I–R group. Values are expressed as the mean ± standard deviation. (*P = 0.043)