Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans

Abstract

Objective: To determine whether ischemic preconditioning protects the human liver against a subsequent period of ischemia in patients undergoing hemihepatectomy, and to identify possible underlying protective mechanisms of ischemic preconditioning, such as inhibition of hepatocellular apoptosis.

Summary background data: Ischemic preconditioning is a short period of ischemia followed by a brief period of reperfusion before a sustained ischemic insult. Recent studies in rodents suggest that ischemic preconditioning is a simple and powerful protective modality against ischemic injury of the liver. The underlying mechanisms are thought to be related to downregulation of the apoptotic pathway.

Methods: Twenty-four patients undergoing hemihepatectomy for various reasons alternatively received ischemic preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) before transection of the liver performed under inflow occlusion for exactly 30 minutes. Liver wedge and Tru-cut biopsy samples were obtained at the opening of the abdomen and 30 minutes after the end of the hepatectomy. Serum levels of aspartate transferase, alanine transferase, bilirubin and prothrombin time were determined daily until discharge. Hepatocellular apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase mediated d-UTP nick end-labeling (TUNEL) assay and electron microscopy. Caspase 3 and 8 activities were measured in tissue using specific fluorometric assays.

Results: Serum levels of aspartate transferase and alanine transferase were reduced by more than twofold in patients subjected to ischemic preconditioning versus controls. The analysis of a subgroup of patients with mild to moderate steatosis indicated possible increased protective effects of ischemic preconditioning. In situ TUNEL staining demonstrated a dramatic reduction in the number of apoptotic sinusoidal lining cells in the ischemic preconditioning group. Electron microscopy confirmed features of apoptosis present in control but not in ischemic preconditioning patients. There was no significant difference in caspase 3 and 8 activity when patients with ischemic preconditioning were compared with controls.

Conclusions: Ischemic preconditioning is a simple and effective modality protecting the liver against subsequent prolonged periods of ischemia. This strategy may be a more attractive technique than intermittent inflow occlusion, which is associated with increased blood loss during each period of reperfusion.

Figure 1. Serum aspartate transferase (A) and alanine transferase (B) levels (mean ± standard deviation) 24 hours after surgery were significantly lower in patients subjected to ischemic preconditioning (□) versus controls (▪) (n = 12 in each group; *P < .01 in each respective comparison, Student t test).

Figure 2. In situ TUNEL staining in Tru-cut liver biopsy samples obtained 30 minutes after the prolonged period of ischemia (30 minutes). Controls (A) showed more positive sinusoidal lining cells than patients subjected to ischemic preconditioning (B). No hepatocyte was found to be TUNEL-positive in either group.

Figure 3. Morphometric analysis of sinusoidal lining cell apoptosis as assessed by the in situ TUNEL assay showed significantly more positive cells in the control group (▪) than in patients subjected to ischemic preconditioning (□). (Data expressed as mean count ± standard deviation per high-power field; n = 5 in each group; *P < .01, Student t test).

Figure 4. Electron microscopic examination of a Tru-cut tissue biopsy showing the most common finding seen after 30 minutes of reperfusion in control livers subjected to 30 minutes of inflow occlusion. A rounded sinusoidal endothelial cell with a dramatic increase in the cytoplasmic/nucleus ratio, which appears detached from the sinusoidal plate, is shown (arrow). These features are suggestive of early apoptosis (H, hepatocyte, RBC, red blood cell).