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. 2004 Oct;155(1-2):55-63.
doi: 10.1016/j.jneuroim.2004.06.003.

Complement C3 and C5 Play Critical Roles in Traumatic Brain Cryoinjury: Blocking Effects on Neutrophil Extravasation by C5a Receptor Antagonist

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Free PMC article

Complement C3 and C5 Play Critical Roles in Traumatic Brain Cryoinjury: Blocking Effects on Neutrophil Extravasation by C5a Receptor Antagonist

Diane L Sewell et al. J Neuroimmunol. .
Free PMC article

Abstract

The role of complement components in traumatic brain injury is poorly understood. Here we show that secondary damage after acute cryoinjury is significantly reduced in C3-/- or C5-/- mice or in mice treated with C5a receptor antagonist peptides. Injury sizes and neutrophil extravasation were compared. While neutrophil density increased following traumatic brain injury in wild type (C57BL/6) mice, C3-deficient mice demonstrated lower neutrophil extravasation and injury sizes in the brain. RNase protection assay indicated that C3 contributes to the induction of brain inflammatory mediators, MIF, RANTES (CCL5) and MCP-1 (CCL2). Intracranial C3 injection induced neutrophil extravasation in injured brains of C3-/- mice suggesting locally produced C3 is important in brain inflammation. We show that neutrophil extravasation is significantly reduced in both C5-/- mice and C5a receptor antagonist treated cryoinjured mice suggesting that one of the possible mechanisms of C3 effect on neutrophil extravasation is mediated via downstream complement activation products such as C5a. Our data indicates that complement inhibitors may ameliorate traumatic brain injury.

Figures

Fig. 1
Fig. 1
Tissue morphology and injury size in the brain of C3 deficient mice are less severe following traumatic brain injury. (A) H&E stained sections of C57BL/6 and (B) C57BL/6/C3−/− mouse brains at 7 days after traumatic injury. At day 7, C3−/− injury sites present a significantly smaller area of leukocyte extravasation with total resolution of the central area of acelluar necrosis, compared to the C57BL/6 brain tissues. (*) represents the edge of the injury site. Images were taken at 100 × magnification. (C) Quantitation of injury areas at 1, 4 and 7 days post injury. Ten H&E stained, coronal sections from the center of injuries of each mouse were photographed, and injury area on each slide was quantified using Scion Image as described in Materials and methods. The three largest areas from each injury site were averaged as maximal cross sectional area. Error bars represent standard error from four mice per group.
Fig. 2
Fig. 2
RPA analysis of inflammatory mediators, and chemokine mRNA expression in brains of C57BL/6 and C57BL6/C3−/− mice. (A) Quantitative inflammatory mediator mRNA expression and (B) chemokine mRNA expression in C57BL/6 or in C57BL6/C3−/− mouse brain injury sites at 1 h, 1 day, 4 days and 7 days following traumatic injury. These images are representative of three experiments. Expression of IL-12p35, IL-6, MIF, Rantes, Eotaxin and MCP-1 were normalized to L32 expression and compared between C57BL/6 wild type mouse brains (black bars) and C3−/− mouse brains (white bars) at 5 time points, uninjured, and 1 h, 1 day, 4 days and 7 days post traumatic brain cryoinjury. Expression level of cytokines and chemokines in uninjured C57BL/6 was arbitrarily set to one with fold change from this level displayed throughout the time course.
Fig. 3
Fig. 3
Neutrophil infiltration in the brain of mice following traumatic cryoinjury. (A) Injury sites 1 day post injury from wild type C57BL/6 and C3−/− brains were stained with H&E, upper panels, and an anti-neutrophil antibody, lower panels. Neutrophils, identified by characteristic segmented nuclei on H&E sections and red staining with anti-neutrophil antibody, are distributed throughout the parenchyma of the injury site in wild type mice and are not contained within vessel lumina (left panels). In complement deficient mouse brain injury sites, the parenchyma contain many fewer neutrophils (right panels). Many neutrophils remain in the luminae of large vessels and fail to extravasate into the parenchyma (right panels). (B) Extravascular neutrophil density per square millimeter of injured tissue was calculated and found to be reduced by ~90% in the absence of complement C3 ( p < 0.005 by Student’s t-test). C3 was administered by intra-cerebral injection of purified mouse C3 (20 μg/injection in 20 μl) into the injury site of C3−/− mice at the time of injury and again 2 h before harvest of 1 day post injury tissue punctuation. Intracerebral administration of C3 partially restored neutrophil extravastion into the injury (n = 4 mice/group, error bars indicate S.D.).
Fig. 4
Fig. 4
C5 deficiency and C5aR antagonists protect mice from acute brain injury. Quantification of extravascular neutrophil density within the injury site as an indicator of severity of inflammation was performed using C5 deficient mice and wild type mice treated with C5aR antagonist. The neutrophil density resulting from the administration of C5aR antagonist prior to cryoinjury of wild type mice (right) was not significantly different from the C5−/− (*p>0.8). In both C5−/− and C5aR antagonist peptide injected wild type mice, extravascular neutrophil density was higher than in C3−/− animals ( p < 0.03, n = 9, mean ± S.D.).

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