Novel in vitro model for studying hepatic ischemia-reperfusion injury using liver cubes

Abstract

Background: Although inflow occlusion techniques have given surgeons the ability to carry out increasingly complex liver resections, ischemia-reperfusion (IR) injury continues to be a source of morbidity. Efforts to ameliorate IR injury have been hindered in absence of adequate preclinical models. The goal of the present study was to develop a simple, efficient, and cost-effective means of studying hepatic IR injury.

Methods: Liver cubes were procured from normal (C57BL/6) mice. After hepatectomy, 4-mm punch biopsies were taken for individual placement in culture wells containing hepatocyte media. Experimental cubes underwent hypoxia for 60 minutes, whereas controls remained normoxic. Supernatants were collected from individual wells after 0, 6, and 12 hours of rediffusion for transaminase and cytokine measurement. Histologic examination was performed on individual cubes.

Results: Extensive histologic injury was seen in the experimental cubes compared with controls with greater staining for activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling at 6 and 24 hours, respectively. Changes consistent with ischemic injury occurred more centrally in liver cubes, whereas markers for rediffusion injury were appreciated along the periphery. Transaminases were significantly higher at 6 hours after rediffusion in experimental cubes compared with controls (P = .02). tumor necrosis factor-α and interleukin-1β were significantly higher in the media of experimental cubes compared with controls at 12 hours rediffusion (P = .05 and .03 respectively).

Conclusion: In vitro IR of cubes produces a significant injury with a pattern reflective of hepatic lobular architecture. This novel technique may open new avenues for uncoupling the mechanisms of IR while facilitating rapid screening of potential therapies.

Figure 1. Liver Cube Procurement and Culture

Left- A 4mm standard punch biopsy tool was utilized to procure liver cubes following hepatectomy. Right- Liver cubes were individually cultured in wells containing 1 ml of hepatocyte basal medium.

Figure 2. Distribution of Injury Following In Vitro ischemia

Following 60 minutes of ischemia, H&E stain reveals a centralized pattern of injury. There is peripheral preservation of cytoarchitecture relative to the core. Images are at 4x magnification.

Figure 3. Distribution of Injury Following In Vitro Rediffusion

Following 6 hours of rediffusion, there is a peripheral pattern of apoptosis. Both activated caspase-3 and TUNEL stain with greater intensity toward the periphery of liver cubes subjected to IR. This pattern of rediffusion injury is in contrast to the centralization of ischemic injury. Lines / arrows have been superimposed to demarcate the peripheral pattern of injury. Images are at 4x magnification.

Figure 4. Comparison of H&E Staining at 6 Hours of Rediffusion

Extensive vacuolar degeneration of an experimental liver cube (A) is seen relative to control (B), whose cytoarchitecture remains intact. Background images are at 10x magnification, whereas the inset is at 40x.

Figure 5. Activated Caspase-3 Staining at 6 hours of Rediffusion

Activity for caspase-3 is considerably greater in an experimental liver cube (A) compared to control (B). Background images are at 10x magnification, whereas the inset is at 40x.

Figure 6. TUNEL Staining at 24 Hours of Rediffusion

TUNEL activity is seen with greater intensity in an experimental liver cube (A) compared to control (B). Background images are at 10x magnification, whereas the inset is at 40x.

Figure 7. Transaminases are Increased Following In Vitro IR of Liver Cubes

Following 60 minutes of either ischemia (experimental) or normoxia (control), there are no differences in media transaminase levels of liver cubes. At 6 hours of rediffusion, AST levels in the media of experimental liver cubes are significantly higher than in controls, p = 0.02.

Figure 8. TNF-α is Increased Following In Vitro IR of Liver Cubes

At 12 hours of rediffusion, media levels of TNF-α are significantly higher in experimental liver cubes than controls, p = 0.05.

Figure 9. IL-1β is Increased Following In Vitro IR of Liver Cubes

At 12 hours of rediffusion, media levels of IL-1β are significantly higher in experimental liver cubes than controls, p = 0.03.