Immunohistochemical Evaluation after Ex Vivo Perfusion of Rectus Abdominis Muscle Flaps in a Porcine Model

Dragu, Adrian; Kleinmann, Judith-Amélie; Taeger, Christian D.; Birkholz, Torsten; Schmidt, Joachim; Geppert, Carol I.; Präbst, Konstantin; Unglaub, Frank; Münch, Frank; Weyand, Michael; Kneser, Ulrich; Horch, Raymund E.

doi:10.1097/prs.0b013e3182589c2d

Cited by 15 publications

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“…In this context, not only organ preservation, but also extracorporeal tissue conservation has made enormous progress. This was demonstrated in case of extracorporeally conserved amputated extremities as well as organs like hearts and lungs . Although interesting prospects may also be seen through further advancement of tissue engineering (TE) and regenerative medicine (RM), at this time TE and RM are not clinically available and hence not yet applicable to the human patients to replace the need of human organs and tissue .…”

Section: Discussionmentioning

confidence: 99%

Ischaemia‐related cell damage in extracorporeal preserved tissue – new findings with a novel perfusion model

Taeger

Müller‐Seubert

Horch

et al. 2014

J Cellular Molecular Medi

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Tissue undergoing free transfer in transplant or reconstructive surgery always is at high risk of ischaemia-related cell damage. This study aims at assessing different procedures using an extracorporeal perfusion and oxygenation system to investigate the expression of hypoxia inducible factor (HIF)-1-α as marker for hypoxia and of the pro-apoptotic protein Caspase-3 in skeletal muscle to elucidate potential improvements in tissue conservation. Twenty-four porcine rectus abdominis muscles were assigned to five different groups and examined after they had been extracorporeally preserved for 60 min. time. Group I was left untreated (control), group II was perfused with a cardioplegic solution, group III was flushed with 10 ml of a cardioplegic solution and then left untreated. Group IV and V were perfused and oxygenated with either an isotone crystalloid solution or a cardioplegic solution. Among others, immunohistochemistry (Caspase-3 and HIF-1-α) of muscle samples was performed. Furthermore, oxygen partial pressure in the perfusate at the arterial and venous branch was measured. Expression of Caspase-3 after 60 min. was reduced in all groups compared to the control group. Furthermore, all groups (except group III) expressed less HIF-1-α than the control group. Oxygenation leads to higher oxygen levels at the venous branch compared to groups without oxygenation. Using an extracorporeal perfusion and oxygenation system cell damage could be reduced as indicated by stabilized expressions of Caspase-3 and HIF-1-α for 60 min. of tissue preservation. Complete depletion of oxygen at the venous branch can be prevented by oxygenation of the perfusate with ambient air.

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Section: Discussionmentioning

confidence: 99%

Ischaemia‐related cell damage in extracorporeal preserved tissue – new findings with a novel perfusion model

Taeger

Müller‐Seubert

Horch

et al. 2014

J Cellular Molecular Medi

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“…HIF-1α could accumulate in the nuclei of the hypoxic cells and the protein can be detected by immunohistochemistry staining. In our previous study, the loop sealed in the isolation chamber was discovered to undergo hypoxia, characterized by HIF-1α up-regulation [ 29 ]. The vascularization followed the increasing of HIF-1α temporally, while spatially it was positively correlated with the HIF-1α level, as well (Data not shown).…”

Section: Introductionmentioning

confidence: 99%

PHDs inhibitor DMOG promotes the vascularization process in the AV loop by HIF-1a up-regulation and the preliminary discussion on its kinetics in rat

et al. 2014

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BackgroundThe Arterovenous Loop (AV Loop) model is a vascularization model in tissue engineering research, which is capable of generating a three dimensional in vivo unit with cells as well as the supporting vessels within an isolation chmaber. In our previous studies the AV loop in the isolation chamber was discovered to undergo hypoxia, characterized by Hypoxia Inducible Factor (HIF) up-regulation. The vascularization followed the increase of HIF-α temporally, while it was spatially positively correlated with the HIF-α level, as well. This study aims to prove that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model.MethodThe AV loop model in rats was created by interposing a femoral vein graft into the distal ends of the contralateral femoral artery and vein, and the loop was embeded in fibrin matrix and fixed in isolation chamber. PHD (prolyl hydroxylases) inhibitor DMOG (Dimethyloxallyl Glycine) was applied systemically in the rats in 40 mg/KG at day 0 and day 3 (DMOG-1), or in 15 mg/KG at day 8, day10 and day12 (DMOG-2). Two weeks later the specimens were explanted and underwent morphological and molecular evaluations.ResultsCompared to the control group, in the DMOG-2 group the HIF-1α positive rate was siginicantly raised as shown in immunohistochemistry staining, accompanied with a smaller cross section area and greater vessel density, and a HIF-1α accumulation in the kidney. The mRNA of HIF-1α and its angiogenic target gene all increased in different extends. Ki67 IHC demostrate more positive cells. There were no significant change in the DMOG-1 group.ConclusionBy applying DMOG systemically, HIF-1α was up-regulated at the protein level and at the mRNA level, acompanied with angiogenic target gene up-regulateion, and the vascularization was promoted correspondingly. DMOG given at lower dosage constantly after one week tends to have better effect than the group given at larger dosage in the early stage in this model, and promotes cell proliferation, as evidenced by Ki67 IHC. Thus, this study proves that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model and that the process of the vessel outgrowth can be controlled in the AV Loop model utilizing this mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0112-x) contains supplementary material, which is available to authorized users.

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“…Second, clotting is a permanent threat and the operation costs are immense. Furthermore, the use of whole autologous blood did not prove feasible as it did not show any superiority compared to Jonosteril in a one hour perfusion setting in a previous experimental setting 18 . Based on the results of Constantinescu’s research, a method was introduced perfusing isolated muscle transplants 36 with alternative perfusate substitutes, including heparinized crystalloid fluid and heparinized cardioplegic solution 22 with promising results.…”

Section: Discussionmentioning

confidence: 92%

“…Both rectus abdominis muscles with the inferior epigastric arteries and veins as pedicle were harvested. The low level of anatomical variation enables uniform operation procedures and the pedicle’s length allows good handling during perfusion 18 36 .…”

Section: Methodsmentioning

confidence: 99%

“…To assess the efficacy of developed conservation protocols, numerous methods have been developed to evaluate tissue viability, functionality or survival probability. Most are located in the field of histology 4 5 8 11 17 and immunohistochemistry 5 9 11 17 18 . The vast majority of these methods demands complex, costly and time-consuming protocols and delivers narrow, localised results from a retrospective point of view.…”

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Assessing viability of extracorporeal preserved muscle transplants using external field stimulation: a novel tool to improve methods prolonging bridge-to-transplantation time

Taeger

Friedrich

Dragu

et al. 2015

Sci Rep

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Preventing ischemia-related cell damage is a priority when preserving tissue for transplantation. Perfusion protocols have been established for a variety of applications and proven to be superior to procedures used in clinical routine. Extracorporeal perfusion of muscle tissue though cumbersome is highly desirable since it is highly susceptible to ischemia-related damage. To show the efficacy of different perfusion protocols external field stimulation can be used to immediately visualize improvement or deterioration of the tissue during active and running perfusion protocols. This method has been used to show the superiority of extracorporeal perfusion using porcine rectus abdominis muscles perfused with heparinized saline solution. Perfused muscles showed statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. The combination of extracorporeal perfusion and external field stimulation may improve organ conservation research.

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Immunohistochemical Evaluation after Ex Vivo Perfusion of Rectus Abdominis Muscle Flaps in a Porcine Model

Abstract: The data of this study indicate that the extracorporal perfusion of muscle flaps with crystalloid fluid is a possible protective strategy against ischemia. Autologous heparinized whole blood seems to have no additional protective effect in a pure perfusion setting without oxygenation.

Cited by 15 publications

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Ischaemia‐related cell damage in extracorporeal preserved tissue – new findings with a novel perfusion model

Ischaemia‐related cell damage in extracorporeal preserved tissue – new findings with a novel perfusion model

PHDs inhibitor DMOG promotes the vascularization process in the AV loop by HIF-1a up-regulation and the preliminary discussion on its kinetics in rat

Assessing viability of extracorporeal preserved muscle transplants using external field stimulation: a novel tool to improve methods prolonging bridge-to-transplantation time

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