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


CONCLUSIONS
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.
Department of Surgery, Washington University in Saint Louis, Saint Louis, MO, USA.
Bernard J
BJ
DuBray
Kendra D
KD
Conzen
Gundumi A
GA
Upadhya
Parvathi
P
Balachandran
Jianluo
J
Jia
Brett L
BL
Knolhoff
David H
DH
Alpers
Thallachallour
T
Mohanakumar
William C
WC
Chapman
Christopher D
CD
Anderson
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D008099:Liver / Q000201:enzymology / Q000473:pathology; D008107:Liver Diseases / Q000201:enzymology / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D015427:Reperfusion Injury / Q000201:enzymology / Q000473:pathology; D046509:Tissue Culture Techniques; D000637:Transaminases / Q000378:metabolism
D016207:Cytokines; D000637:Transaminases; D053148:Caspase 3