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«Die protektive Wirkung von Cyclosporin A während der Ischämie und der Reperfusion Untersuchungen am global ischämischen Hundeherzen ...»

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Zusammenfassend bestätigen die Ergebnisse eine deutliche protektive Wirkung von CsA, die in Kombination mit Hypothermie und UW-Lösung zu einer drastischen Verbesserung der funktionellen und strukturellen Erholung nach Langzeitischämie und Reperfusion führt.

Dabei verringert CsA einerseits die in der CsA-freien Gruppe auftretenden ischämischen Beschädigungen, andererseits reduziert CsA signifikant die im Verlauf der Ischämie eingeleitete Apoptose.

Beide Effekte werden maßgeblich durch die CsA-bedingte Hemmung der Ca2+abhängigen mitochondrialen „permeability transition“-Pore verursacht. Dadurch verhindert CsA die Schwellung der Mitochondrien und die Freisetzung des Apoptoseinduktors Cytochrom C. Außerdem blockiert CsA die pro-apoptotische Wirkung von Calcineurin durch die vollständige Enzymdeaktivierung.

Als CsA-unabhängig erwiesen sich die Proteine der Bcl-2-Familie. In beiden Versuchsgruppen änderte sich das Konzentrationsverhältnis zwischen den pro- und anti-apoptotischen Mitgliedern der Familie zugunsten des anti-apoptotischen Bcl–2‘s. Hieraus resultiert vermutlich die geringe Apoptoserate trotz der 18stündigen Ischämie.

Insgesamt deuten die Ergebnisse auf eine herausragende Rolle von CsA bei der Entwicklung neuer kardioplegischer Lösungen für die Herzaufbewahrung bei humanen Transplantationen hin, wobei neben der Vergrößerung des Donorpools vor allem die schnellere Erholung des Organs von Bedeutung wäre.

Inwiefern andere Mechanismen (z.B. IAP oder NO) an der Verbesserung der Ischämietoleranz beteiligt sind, muss in zukünftigen Versuchen untersucht werden.

VII. Summary - 109 VII. SUMMARY Cyclosporine A, an immunosuppressive cyclic undecapeptide initially isolated from Tolypocladium inflatum gams, has been widely used for the treatment of allograft rejection.

The aim of the study was to determine a possible protective role against ischemia and to clarify the working mechanisms of cyclosporine A (CsA) in a canine cardiac long-term ischemia model. Therefore twelve dog hearts were perfused with „University of Wisconsin“-solution during18 hours of hypothermic global ischemia.

In six hearts, CsA was added to the cardioplegic solution and the others were used as control. After 18 hours, the hearts were transplanted into the recipient dogs to

reestablish blood perfusion. During the following six hours four biopsies were taken:

before transplantation (A), at the end of preservation (B), after two hours (C), and after six hours of reperfusion.

Furthermore, the end-systolic elastance was calculated during the reperfusion period to measure functional recovery with the addition of CsA. The function recovered to 55% in the CsA-free group, whereas recovery increased to 100% in CsA-treated hearts.

To further elucidate the cause for these protective effects histomorphological

investigations were performed with the following results:

The degree of ischemic damage was determined by electron microscopy. Although most of the hearts showed reversible injury, the degree of damage was elevated in the group without CsA.

Electronmicroscopic evidence for necrosis was not observed. This correlates with the absence of positive complement C9 labeling in all biopsies.

In contrast to necrosis, 6% of the total number of myocytes in the untreated group were apoptotic (TUNEL-positive) after 6 hours of reperfusion. This rate significantly decreased to 2,4% when CsA was added to the cardioplegic solution.

These findings were confirmed by Lamin B1-staining where a loss of 7% of Lamin B1 positive nuclei was detected in the D-biopsie of non treated hearts in comparison to 3% in the treated group. Corresponding results were found concerning the number of Lamin A negative nuclei. Their number increased to 27% (without CsA) and to only 5% (with CsA) at the end of the reperfusion period.

To elucidate the working mechanisms of the substance in detail, Western Blot analysis of different proteins was performed. In particular, the quantitative evaluations of mitochondrial cytochrome c showed in a significant difference between both groups. After 18 hours of ischemia, the amount of cytochrome c was

- 110 - VII. Summary reduced to 48% in CsA-free dogs in comparison to 73% in dogs treated with CsA. In contrast, the total amount of Bax showed no significant changes until the end of the reperfusion period. At this time point, the amount of protein was reduced to 65% (without CsA) and 71% (with CsA), respectively. The same decrease after 6 hours of reperfusion was valid for Calcineurin and Bcl-2, whereby the total quantity of Bcl-2 moderately increased during ischemia before reaching 70% without and 81% with the addition of CsA at the end of reperfusion. Calcineurin decreased from 100% to 64% in the CsA free-group and to 72% in CsA perfused hearts.

Another important finding is the fact, that calcineurin activity was completely blocked during ischemia and reperfusion in the CsA-treated group. In comparison, the activity decreased to 70% at the end of the experiment in untreated hearts.

To study a damaging effect of ischemia/reperfusion on structural proteins Western blot-analysis and histomorphology for alpha-actinin and actin were carried out. No differences were found between the total amount of protein at the four time points but fluorescent labeling of alpha-actinin showed areas with diffuse alpha-actinin labeling in the majority of C- and D-biopsies.





In conclusion, these results demonstrate protective effects of CsA in combination with hypothermia and UW-solution which lead to a drastic increase in functional and structural recovery after longterm ischemia and reperfusion.

Moreover, the addition of CsA diminished on the one hand the degree of ischemic damage as well as significantly reduced the rate of apoptotic cells.

Both mechanisms were caused by stabilization of the mitochondrial permeability transition pore through CsA thereby preventing the mitochondrial swelling and the release of the apoptotic inducer cytochrome c. Furthermore, the CsA-induced blockade of calcineurin activity inhibits the pro-apoptotic effects of calcineurin.

The present results prove that the principal working mechanisms of the Bcl-2family are independent of CsA. In both groups, the ratio between the pro-apoptotic and anti-apoptotic-members of this family changed towards the anti-apoptotic Bcl–2. This might be the reason for the occurrence of a small number of apoptotic cells after 18 hours of global ischemia.

In summary this data indicates a potential role for CsA in cardioplegic solutions used in transplantation of human hearts. Consequently, the addition of CsA could expand the donorpool and improve the functional recovery after heart transplantations.

VIII. Abkürzungen - 111

–  –  –

NADH2 Nicotinamid-adenin-dinucleotid (reduzierte Form) NF-AT „nuclear factor of activated T-cell“ NOS Stickstoffmonoxid-Synthase PARP Polyadenylribosepolymerase PBS Phosphat-gepufferte physiologische Kochsalzlösung PFA Paraformaldehyd PMSF Phenyl-Methyl-Sulfonyl-Fluoride PP Protein-Phosphatase PTP „permeability transition“-Pore RT Raumtemperatur Ser Serin SEM Standardfehler TBS Tris-Base-Saline TCA Tri-Chlor-Säure TdT terminale Deoxynucleotidyl-Transferase TG Trockengewicht TNF-alpha Tumor Nekrose Faktor alpha TRADD „TNF-receptor-associated death domain“ TRITC Tetramethylrhodamin-Isothiocyanat TUNEL „terminal-deoxy-nucleotidyl-transferase mediated d-UTP nick-endlabeling UW „University of Wisconsin“ UPM Umdrehungen pro Minute V. Vena

-/- Mäuse „knock out“ Mäuse IX. Literaturverzeichnis - 113 IX. Literaturverzeichnis

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–  –  –

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