Donor brain death exacerbates complement-dependent ischemia/reperfusion injury in transplanted hearts

Abstract

Background: Brain death (BD) can immunologically prime the donor organ and is thought to lead to exacerbated ischemia/reperfusion injury after transplantation. Using a newly developed mouse model of BD, we investigated the effect of donor BD on posttransplantation cardiac ischemia/reperfusion injury. We further investigated the therapeutic effect of a targeted complement inhibitor in recipients of BD donor hearts and addressed the clinical relevance of these studies by analyzing human heart biopsies from BD and domino (living) donors.

Methods and results: Hearts from living or BD donor C57BL/6 mice were transplanted into C57BL/6 or BALB/c recipients. Recipient mice were treated with the complement inhibitor CR2-Crry or vehicle control (n=6). Isografts were analyzed 48 hours after transplantation for injury, inflammation, and complement deposition, and allografts were monitored for graft survival. Human cardiac biopsies were analyzed for complement deposition and inflammatory cell infiltration. In the murine model, donor BD exacerbated ischemia/reperfusion injury and graft rejection, as demonstrated by increased myocardial injury, serum cardiac troponin, cellular infiltration, complement deposition, inflammatory chemokine and cytokine levels, and by decreased graft survival. CR2-Crry treatment of recipients significantly reduced all measured outcomes in grafts from both BD and living donors compared with controls. Analysis of human samples documented the relevance of our experimental findings and revealed exacerbated complement deposition and inflammation in grafts from BD donors compared with grafts from living donors.

Conclusions: BD exacerbates posttransplantation cardiac ischemia/reperfusion injury in mice and humans and decreases survival of mouse allografts. Furthermore, targeted complement inhibition in recipient mice ameliorates BD-exacerbated ischemia/reperfusion injury.

Figure 1

Mean arterial pressures of brain dead heart beating donors over 3 hour period. Isograft (A) and allograft (B) recipients grouped based on their post transplant treatment groups (CR2- Crry therapy or no therapy). General linear mixed models indicated that for both the isograft and allograft experiments, there were no significant differences in mean MAP values over time between the BD and BD+CR2-Crry groups. In isograft experiment, the mean difference between groups MAP values over time was 0.8 mmHg (95% confidence interval: −9.1 to 10.6, p=0.87). In the allograft experiment, the mean difference between groups MAP values over time was 0.7 mmHg (95% confidence interval: −7.2 to 8.7, p=0.83).

Figure 2

Assessment of cardiac injury in recipients of living or brain dead donor hearts. Where indicated, recipients were treated with CR2-Crry. A. Histological quantification of cardiac injury in grafts harvested 48 hrs post transplantation, quantified using 0-12 cumulative injury score. Pairwise comparisons between living vs. living+CR2-Crry (#p<0.05), living vs. BD (**p<0.05) and BD vs. BD+CR2-Crry (##p<0.05). Differences between living vs. BD+CR2- Crry and between living vs. BD Sham were not significant. B. Serum cardiac troponin I levels in recipient mice 48 hrs post transplantation. Pairwise comparisons between living vs. living+CR2- Crry (#p<0.05), living vs. BD (**p<0.001) and BD vs. BD+CR2+Crry (##p<0.001). Differences between living vs. BD+CR2-Crry and living vs BD Sham were not significant. Results are expressed as Mean ± SE; (n=6-12).

Figure 3

C3d deposition in grafts isolated 48hrs post transplantation. A. Representative C3d immunostained images from recipients of living and BD donor cardiac grafts. C3d is localized to endothelial cells and myocytes. Representative of n=6. Original magnification x10. B. Semiquantitative analysis of C3d deposition in grafts from living and BD donors. Pairwise comparisons between living vs. living+CR2-Crry (#p<0.05), living vs. BD (**p<0.01) and BD vs. BD+CR2-Crry (##p<0.001). Differences between living vs. BD+CR2-Crry and living vs BD Sham were not significant. Results expressed as Mean ± SE; (n=6).

Figure 4

Effect of brain death and complement inhibition on inflammatory cell infiltration. A. Myeloperoxidase activity in cardiac grafts 48 hrs post transplantation. Pairwise comparisons between living vs. living+CR2-Crry (#p<0.05), living vs. BD (**p<0.001) and BD vs. BD+CR2- Crry (##p<0.001). Differences between living vs. BD+CR2-Crry and living vs BD Sham were not significant. Immunohistochemistry quantification of neutrophil (B) and macrophage (C) infiltration as marked by GR-1 and MAC-3, respectively. Pairwise comparisons for neutrophil studies (B) between living vs. living+CR2-Crry (#p<0.05) and living vs. BD (**p<0.01). Differences between BD vs. BD+CR2-Crry, living vs. BD+CR2-Crry and Living vs. BD Sham were not significant. For macrophage studies (C), pairwise comparisons between living vs. living+CR2-Crry (#p<0.05), living vs. BD (**p<0.01) and BD vs. BD+CR2-Crry (##p<0.01). Differences between living vs. BD+CR2-Crry and living vs. BD Sham were not significant. Results expressed as Mean ± SE; (n=4-6).

Figure 5

Effect of brain death and complement inhibition on graft chemokine and cytokine expression. Cardiac grafts were harvested 48 hrs post transplantation. Cytokines in cardiac graft homogenates were measured by ELISA. Pairwise comparisons between living vs. living+CR2- Crry (#p<0.05), living vs. BD (**p<0.001, with exception of MCP-1, which was not significant) and BD vs. BD+CR2-Crry (##p<0.001). Differences between living vs. BD+CR2-Crry and living vs. BD Sham were not significant. Results expressed as mean ± SE; (n=4-6).

Figure 6

Effect of brain death and complement inhibition on allograft survival post transplantation. Hearts from C57BL/6 living, sham or BD donors were transplanted into BALB/c recipients treated with either CR2-Crry or PBS. Logrank tests comparing survival across the 4 groups demonstrated survival times were significantly different between groups (p<0.0001). All pairwise comparisons demonstrated that survival was significantly greater among each group when compared to the BD group (p<0.01 for all), even after a Bonferroni adjustment for multiple comparisons. No significant difference was seen between living, Sham BD or BD+CR2-Crry. (n=4-6).

Figure 7

Immunohistochemistry localization of C4d on human cardiac biopsies obtained at donor optimization from living and brain dead donors. Note the capillary immunostaining of endothelial cells in the brain dead samples. Images are representative of n=3 for living, and n=8 for brain dead donors. Original magnification x400.

Figure 8

Immunohistochemistry localization of monocyte/macrophages (MAC387) and mature tissue macrophages (CD68) in human cardiac biopsies from living and BD donor organs. Results expressed as Mean ± SE; n=3-10. #p<0.05 living vs. BD.