Complement-dependent inflammation and injury in a murine model of brain dead donor hearts

Abstract

Rationale: Donor brain death (BD) is an unavoidable occurrence in heart transplantation and results in profound physiological derangements that render the heart more susceptible to ischemia/reperfusion injury in the recipient and likely has negative long-term consequences to allograft survival.

Objective: We developed a novel mouse model of BD and investigated the role of complement in BD-induced myocardial inflammation and injury.

Methods and results: BD was induced by inflation of a balloon catheter in the cranial cavity. BD in wild-type mice resulted in a significant increase in serum concentrations of the complement activation product complement component (C)3a, and immunohistochemical analysis of heart sections demonstrated C3 deposition on the vascular endothelium and surrounding myocytes. Following induction of BD in complement (C3)-deficient mice, cardiac troponin levels, and histological evidence of injury were significantly reduced compared to wild-type mice. C3 deficiency was also associated with reduced myocardial leukocyte infiltration and reduced or absent expression of P-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor-alpha, and interleukin-1beta.

Conclusions: These data indicate an important role for complement in BD-induced inflammation and injury and suggest that a complement inhibitory strategy applied to the donor (in addition to the recipient) may provide graft protection.

Figures

Figure 1

Mean arterial blood pressure traces from wild type (A) and C3−/− (B) mice prior to and following induction of BD. Note that the MAP response to BD is similar in each group of animals. Representative images (n =10).

Figure 2

Complement activation and myocardial injury following brain death. A: Relative levels of complement activation product C3a in serum from wt brain dead animals and sham operated controls after 3 hours of ventilation support, as determined by ELISA (p < 0.01 , n = 6). Results are expressed as a percentage increase in C3a levels when compared to normal mouse serum. B: Representative images of C3d deposition assessed by immunohistochemistry at 3 hours post BD in wt and C3−/− mice. Positive staining for C3d (brown) can bee seen in muscularized vessels within the heart myocardium of wt mice. Staining is absent in hearts from non-brain dead controls and, as expected, in hearts from C3−/− mice, which serve as a negative control. Representative images, (n = 4 for each group). C: Representative images of IgM deposition at 3 hrs post BD in wt and C3−/− mice. Positive staining for IgM (Brown) can be seen on the endothelium of muscularized vessels and capillaries in both wt and C3−/− animals. Some diffuse staining can be seen on myocytes in both groups. Representative images, (n = 4 for each group).

Figure 3

Cardiac damage following brain death. A. Serum levels of cardiac troponin I in wild type and C3−/− mice following BD. Cardiac troponin levels were analyzed by ELISA in serum samples prepared from 3 hour brain dead animals. The data indicate reduced myocyte injury in C3−/− mice compared to wt. Mean ± SD (p=0.03, n = 8). B. Representative hematoxylin and eosin stained sections of wt and C3−/− mice 3 hours post BD induction. Note the presence of endothelial activation as denoted by endothelial swelling and prominent endothelial nuclei in wt animals as compared to the flatten nuclei profile seen in C3−/− mice. Images representative of n=8 in each group, images taken under oil immersion.

Figure 4

Adhesion molecule protein expression in wild type and C3 −/− mice following brain death. Expression of P-selectin, I-CAM-1, V-CAM and E-selectin was examined in hearts from wt and C3−/− mice at 3 hours following BD. Note reduced expression of all adhesion molecules in C3−/− mice compared to wt. Staining intensities are quantified in Supplemental Table II. Representative images (n = 6 for each group.)

Figure 5

Transcript levels of adhesion molecules and cytokines in wild type and C3−/− mice following brain death. mRNA levels were determined in heart samples isolated from mice 3 hours post BD by quantitative RT-PCR. A: Adhesion molecule transcript expression. V-CAM-1 and E-selectin gene transcription was significantly increased in hearts from wt mice compared to hearts from C3−/− mice (p < 0.02, n = 6). B: TNF-α and IL-1β transcript expression. There was a significant increase in gene transcription for both cytokines in hearts from wt mice compared to hearts from C3−/− mice (p < 0.05, n = 6).

Figure 6

Neutrophil infiltration in hearts from wt and C3−/− mice following BD. Representative images for wt (A) and C3−/− (B) immunostained with GR-1 for neutrophil localization. C & D. Quantification of neutrophil staining, immunohistochemistry with GR-1 (C) and enzyme histochemistry with naphthol AS-D chloroacetate esterase (D), shows a low numbers of neutrophils in hearts post brain death. However, wt hearts have a significant increase in total neutrophils/5HPF. (n=10, p