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Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model
Published on: April 2, 2017
Effect of activation on neutrophil-induced hepatic microvascular injury in isolated rat liver
J X Zhang1, D V Jones, M G Clemens
1Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Abstract:
Polymorphonuclear neutrophils (PMNs) have been implicated in microvascular injury following ischemia and reperfusion (I/R) but the relative contribution of obstruction versus toxic mediators is not well defined. Therefore, the present study was performed to determine the contribution of exogenous or endogenous activation on PMN-induced microvascular and hepatocyte injury. Rat livers were isolated and perfused at constant pressure with Krebs buffer with red cells (Hct-10%) and monitored for perfused sinusoids (PS) and dead hepatocytes (propidium iodide-stained, DH) by intravital microscopy. PMNs isolated from the peritoneum after oyster glycogen injection were added to the perfusate either without or with activation by phorbol myristate acetate (PMA, 160 nM). Unactivated PMNs stuck in the liver but had no significant effect on either perfused sinusoids (11.1 +/- .4/field, unactivated PMNs versus 11.9 +/- .5/field, the time-matched control) or dead hepatocytes (1.2 +/- .4/field, unactivated PMNs versus 1 +/- .3/field, the time-matched control). Infusion of PMA-activated PMNs resulted in significant decrease in perfused sinusoids and increase in DH (9.5 +/- .3/field for PS and 3.2 +/- .6/field for DH, respectively). In contrast, when PMNs were "activated" by infusion into a liver previously made ischemic for 30 min, DH were significantly increased after 60 min (26.2 +/- 4.5/field, I/R plus PMNs versus 12.4 +/- 2/field, I/R only) but perfused sinusoids were not different from ischemia alone. These results demonstrate that oxidatively quiescent PMNs do not cause cellular or microvascular injury in spite of microvascular accumulation. Activated PMNs damage microcirculation or hepatocytes depending on the nature of the activation.
Insights
Activated polymorphonuclear neutrophils (PMNs) cause liver injury, but unactivated PMNs do not. The type of PMN activation determines whether microvascular or hepatocyte damage occurs during ischemia-reperfusion.
Area of Science:
- Hepatology
- Immunology
- Microcirculation Research
Background:
- Polymorphonuclear neutrophils (PMNs) are implicated in microvascular injury following ischemia and reperfusion (I/R).
- The specific mechanisms, whether obstruction or toxic mediators, driving PMN-induced injury remain unclear.
Purpose of the Study:
- To investigate the distinct contributions of exogenous versus endogenous PMN activation to liver injury.
- To differentiate the effects of activated versus unactivated PMNs on microvascular and hepatocyte damage in an ex vivo rat liver model.
Main Methods:
- Isolated rat livers were perfused with Krebs buffer containing red blood cells.
- Intravital microscopy monitored perfused sinusoids and dead hepatocytes.
- PMNs were added to the perfusate, either unactivated or activated with phorbol myristate acetate (PMA), or infused into ischemic livers.
Main Results:
- Unactivated PMNs accumulated in the liver but did not significantly affect sinusoid perfusion or hepatocyte death.
- PMA-activated PMNs significantly reduced sinusoid perfusion and increased hepatocyte death.
- PMNs infused into ischemic livers increased hepatocyte death but did not alter sinusoid perfusion compared to ischemia alone.
Conclusions:
- Oxidatively quiescent PMNs do not cause significant injury despite microvascular accumulation.
- PMN activation is critical for inducing microvascular and hepatocyte injury during I/R.
- The nature of PMN activation dictates whether microcirculatory or direct hepatocyte damage predominates.
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