|Title:||The development of an in vitro model of hypoxia reoxygenation in precision-cut liver slices for studying the effects of nitrite in hypoxic liver tissue||Language:||English||Authors:||Ghebes, Corina-Adriana||Keywords:||Hypoxie; Reoxygenierung; Leber Schnitte; Nitrit
Hypoxia; Reoxygenation; Precision-cut liver slices; Nitrite
|Advisor:||Redl, Heinz||Assisting Advisor:||Kozlov , Andrey||Issue Date:||2011||Number of Pages:||49||Qualification level:||Diploma||Abstract:||
Hepatic ischemia/reperfusion (I/R) induced injury is a significant medical problem in many clinical conditions such as liver transplantation, hepatic surgery for tumor excision, trauma and hepatic failure after hemorrhagic shock. I/R injury is characterized by release of tissue enzyme, mitochondria damage, increase of reactive oxygen species (ROS) generation, oxidative modification of essential proteins and lipids, etc.
Recently, it was shown that nitrite can reduce I/R injury due to its ability to be reduced to NO during ischemia and limiting hence cytotoxicity and apoptosis at reperfusion. NO has been demonstrated to inhibit oxidative stress, to inhibit mitochondria respiration (complex I) and to limit apoptotic cytochrome c release, however the exact mechanism is still unknown.
Therefore, to investigate the mechanism of nitrite mediated cytoprotection in I/R induced injury a proper in vitro model is needed.
Precision-cut liver slices (PCLS), used for in vitro investigations, has gained more and more attention in the last years due to their property to retain tissue organization, to include different cell types and to maintain cell to cell matrix interactions, in contrast to cell culture model.
The aim of this study was first to establish a hypoxia/reoxygenation model using PCLSs and second to investigate the effects of nitrite treatment on cell function and survival upon hypoxia/reoxygenation.
PCLSs were prepared from rat liver using a Compresstome VF-300 Instrument and incubated for one hour under hypoxia and for another hour under reoxygenation conditions, in glass vials containing incubation medium. At the beginning of the hypoxia incubation, different concentrations of nitrite were added in the incubation medium. For the investigation of pathophysiological changes occurred in the tissue as result of incubation conditions, we analyzed different parameters such as: mitochondria respiratory function, measured with the Oroboros Oxygraph-2k Instrument; liver damage by AST, ALT and LDH release, measured with the auto-analyser Cobas c 111 and RNA quality and gene expression, measured with Agilent 2100 Bioanalyser and iCycler iQ Bio-Rad.
Additionally to one hour hypoxia and one hour reoxygenation incubation, we performed two more sets of experiments. For the first set, we used the same model but investigating two hours of hypoxia or half an hour hypoxia and one and a half hour reoxygenation in the presence/absence of glucose. For the second set, we designed a new model that simulates hypoxia/reoxygenation directly in the Oroboros respirometer chambers, allowing hence an online measurement of mitochondria respiration in PCLSs in the presence of nitrite.
The results obtained are as followed: hypoxia/reoxygenation simulation on PCLSs led to a partial or total inhibition of mitochondria respiration, to a release in AST and ALT enzymes, to highly reduced mRNA amounts and to an up-regulation of HO-1. Effects of nitrite were not identified in the one hour hypoxia and one hour reoxygenation model.
However nitrite effects were visible in the online measurement of mitochondria respiration during hypoxia/reoxygenation, where nitrite led to a decrease in mitochondria respiration during hypoxia. Regarding glucose, we observed an impair effect of glucose on the tissue during the reoxygenation.
Taken together, our model demonstrated a good simulation of hypoxia/reoxygenation conditions, however probably too aggressive for the tissue and consequently no effects of nitrite were visible.
Therefore I suggest, for further investigations of nitrite effects on hypoxia/reoxygenation induced injury in PCLSs, to reduce the incubation duration, especially that of reoxygenation, as observed to have a major impair effect on the tissue.
|Library ID:||AC07812058||Organisation:||E166 - Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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