Oxidative stress and apoptosis in a pig model of brain death (BD) and living donation (LD)

Abstract

Background: As organ shortage is increasing, the acceptance of marginal donors increases, which might result in poor organ function and patient survival. Mostly, organ damage is caused during brain death (BD), cold ischemic time (CIT) or after reperfusion due to oxidative stress or the induction of apoptosis. The aim of this study was to study a panel of genes involved in oxidative stress and apoptosis and compare these findings with immunohistochemistry from a BD and living donation (LD) pig model and after cold ischemia time (CIT).

Methods: BD was induced in pigs; after 12 h organ retrieval was performed; heart, liver and kidney tissue specimens were collected in the BD (n = 6) and in a LD model (n = 6). PCR analysis for NFKB1, GSS, SOD2, PPAR-alpha, OXSR1, BAX, BCL2L1, and HSP 70.2 was performed and immunohistochemistry used to show apoptosis and nitrosative stress induced cell damage.

Results: In heart tissue of BD BAX, BCL2L1 and HSP 70.2 increased significantly after CIT. Only SOD2 was over-expressed after CIT in BD liver tissue. In kidney tissue, BCL2L1, NFKB, OXSR1, SOD2 and HSP 70.2 expression was significantly elevated in LD. Immunohistochemistry showed a significant increase in activated Caspase 3 and nitrotyrosine positive cells after CIT in BD in liver and in kidney tissue but not in heart tissue.

Conclusion: The up-regulation of protective and apoptotic genes seems to be divergent in the different organs in the BD and LD setting; however, immunohistochemistry revealed more apoptotic and nitrotyrosine positive cells in the BD setting in liver and kidney tissue whereas in heart tissue both BD and LD showed an increase.

Figure 1

Apoptosis and oxidative stress related gene expression in pig heart tissue. A) LD heart apoptosis genes, B) BD heart apoptosis genes, C) LD heart oxidative stress genes, D) BD heart oxidative stress genes *p < 0.05, ** p < 0.01, ***p < 0.001; BD: Brain death; LD: Living donation; CIT: cold ischemic time.

Figure 2

For immunohistochemistry, an antibody against activated human/mouse Caspase 3 (dilution 1:50) was used for the detection of apoptosis in the different tissues at different time-points. Heart: A, B: Representative stainings for heart tissue of BD prior to perfusion (A) and after 4 h of CIT (B) against activated Caspase 3. C, D: Representative stainings of nitrotyrosine positive cells in BD hearts prior to perfusion (C) and after 6 h of CIT (D). Nitrotyrosine positive cells were more common in BD hearts after CIT, at all other time points no difference between LD and BD was found. Liver: E, F: Representative stainings of liver tissue of BD prior to perfusion (E) and after 6 h of CIT (F) against activated Caspase 3. G, H: Representative stainings for nitrotyrosine positive cells of BD liver tissue prior to perfusion (G) and after 6 h of CIT (H). Kidney: I, J: Representative stainings for kidney tissue against activated Caspase 3 prior to perfusion (I) and after 15 h of CIT (J). In kidney tissue, the number of activated Caspase 3 positive cells was significantly higher in both groups after 15 h CIT compared to prior to perfusion and was higher in BD donor organs compared to LD organs after CIT. Especially tubular tissue seemed to be more susceptible to occurrence of apoptosis after CIT. K, L: Staining for nitrotyrosine positive cells in BD kidney tissue prior to perfusion (K) and after 15 h of CIT (L). Tubular cells seemed to be more affected by nitrosative stress in BD kidney tissue after CIT. 100 x magnification. The inserts show areas of interest 200 x magnification. Arrow heads: examples of activated Caspase 3 positive cells (A, B, E, F, I, J) and nitrotyrosine positive cells (C, D, G, H, K, L); CIT: Cold ischemic time.

Figure 3

Apoptosis and oxidative stress related gene expression in pig liver tissue. A) LD liver apoptosis genes, B) BD liver apoptosis genes, C) LD liver oxidative stress genes, D) BD liver oxidative stress genes *p < 0.05, ** p < 0.01, ***p < 0.001; BD: Brain death; LD: Living donation; CIT: cold ischemic time.

Figure 4

Apoptosis and oxidative stress related gene expression in pig kidney tissue. A) LD kidney apoptosis genes, B) BD kidney apoptosis genes, C) LD kidney oxidative stress genes, D) BD kidney oxidative stress genes *p < 0.05, ** p < 0.01, ***p < 0.001 BD: Brain death; LD: Living donation; CIT: cold ischemic time.