Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death

Abstract

Stroke is among the three leading causes of death worldwide and the most frequent cause of permanent disability. Brain ischemia induces an inflammatory response involving activated complement fragments. Here we show that i.v. Ig (IVIG) treatment, which scavenges complement fragments, protects brain cells against the deleterious effects of experimental ischemia and reperfusion (I/R) and prevents I/R-induced mortality in mice. Animals administered IVIG either 30 min before ischemia or after 3 h of reperfusion exhibited a 50-60% reduction of brain infarct size and a 2- to 3-fold improvement of the functional outcome. Even a single low dose of IVIG given after stroke was effective. IVIG was protective in the nonreperfusion model of murine stroke as well and did not exert any peripheral effects. Human IgG as well as intrinsic murine C3 levels were significantly higher in the infarcted brain region compared with the noninjured side, and their physical association was demonstrated by immuno-coprecipitation. C5-deficient mice were significantly protected from I/R injury compared with their wild-type littermates. Exposure of cultured neurons to oxygen/glucose deprivation resulted in increased levels of C3 associated with activation of caspase 3, a marker of apoptosis; both signals were attenuated with IVIG treatment. Our data suggest a major role for complement-mediated cell death in ischemic brain injury and the prospect of using IVIG in relatively low doses as an interventional therapy for stroke.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

IVIG treatment reduces brain damage and improves functional outcome in a mouse stroke model. (a) A five-point neurological score was applied to the sham (n = 5), vehicle-treated I/R (n = 12), albumin-treated I/R (n = 7), and IVIG- (2 g/kg) pretreated I/R (n = 10) mice; *, P < 0.001 compared with sham value; +, P < 0.01 compared with vehicle treated I/R value. (b) Ischemic infarct sizes 72 h after I/R in vehicle-treated (n = 12), I/R albumin-treated (n = 7), and IVIG-pretreated (n = 10) mice; *, P < 0.001 compared with sham value; +, P < 0.01 compared with vehicle-treated I/R value. (c) Neurological function in mice posttreated with 2 g/kg IVIG either 1 h (n = 8) or 3 h (n = 8) after ischemia or with 0.5 g/kg IVIG 1 h after ischemia (n = 9) and albumin at 2 g/kg (n = 10) 3 h into reperfusion; +, P < 0.05 compared with vehicle-treated mice (n = 8); *, P < 0.01 compared with sham-operated mice (n = 10). (d) The infarct size was significantly reduced in mice treated with 2 g/kg IVIG 3 h (n = 8) or 1 h (n = 7) after stroke as well as with 0.5 g/kg IVIG 1 h after stroke (n = 8) but not in mice treated with 2 g/kg albumin (n = 10) compared with vehicle-treated mice (n = 8); *, P < 0.001 compared with sham value; +, P < 0.05 compared with vehicle-treated mice. (e) Representative images (from brain sections taken from four animals in each treatment group) of 2,3,5-triphenyltetrazolium chloride-stained sections (Top) and hematoxylin/eosin-stained sections at ×4 (Middle) and ×20 (Bottom) magnification from both vehicle-treated I/R mice and mice treated with 2 g/kg IVIG 1 h after stroke. (f) Infarct volume in serial brain sections from mice treated with vehicle (n = 8), IVIG (n = 8), and albumin (n = 10) 30 min before being subjected to PMCAO; +, P < 0.05; *, P < 0.01. (g) The effect of vehicle (n = 8), albumin (n = 9), and IVIG (n = 8) on brain infarct size after administration 1 h after PMCAO; +, P < 0.05; *, P < 0.01. (h) The effect 3 h posttreatment with vehicle (n = 6), albumin (n = 8), and IVIG (n = 10) on infarction size in PMCAO; +, P < 0.05; *, P < 0.01.

Fig. 2.

The integrity of the BBB is compromised in ischemic brain regions. (a) Staining for human IgG (green fluorescence) in brain sections from the site of infarction and the corresponding site from nonaffected hemisphere. (Scale bars: 100 μM.) (b) Double staining for collagen IV, blood vessel marker (red fluorescence), and human IgG (green fluorescence) reveals the presence of human IgG in both blood vessels and parenchyma at the site of injury and intravascular localization in the contralateral side. (c) Double staining for glial cell marker IBA1 (red fluorescence) and human IgG (green fluorescence) and their colocalization in the ipsilateral side.

Fig. 3.

Contribution of C5 and C3 complement components to pathophysiology of experimental stroke and attenuation with IVIG. Neurological scores (a) and infarct sizes (b) 24 h after I/R brain injury in wild-type (C5+/+) sham-operated mice (n = 8) and the following groups of mice subjected to I/R stroke: C5+/+ mice (n = 10), C5−/− mice (n = 10), vehicle-treated C5+/+ mice (n = 12), C5a receptor antagonist (C5aRA)-treated C5+/+ mice (n = 8), and IVIG-treated C5+/+ mice (n = 10). C5aRA and IVIG were administered 30 min before onset of ischemia; *, P < 0.001 compared with the sham value; +, P < 0.05 compared with the C5+/+ I/R value. (c) Proteins in cerebral cortex samples from mice subjected to sham surgery (n = 3), vehicle I/R (n = 4), and IVIG-treated at 2 g/kg administered 1 h after stroke (n = 4) were immunoblotted by using anti-C3b and antiactin antibodies. (d) C3b levels determined by densitometric analysis of immunoblots (normalized to actin levels and expressed as a percentage of the mean value for sham-treated mice) in infarcted brain region compared with sham (*, P < 0.01) and vehicle-treated (+, P < 0.01) controls. Quantification of C3b levels (e) and immunoblots (g) in the ischemic area as compared with the corresponding contralateral area; *, P < 0.01 compared with the sham value; +, P < 0.01 compared with the vehicle I/R value. (f) Staining for C3 (red membranous fluorescence), neuronal nuclear marker NeuN (green fluorescence), and double staining including nonneuronal nuclear staining (DAPI) show expression of intrinsic neuronal C3 in the infarcted hemisphere (arrows). (h) Coimmunoprecipitation of human IgG and mouse C3 in the infarcted brain sample.

Fig. 4.

IVIG treatment suppresses endothelial cell adhesion, lymphocyte infiltration, and microglial activation after a stroke. Representative images of CD11a (a) and CD11b (b) immunoreactivity and corresponding quantitative analysis (c and d) in the indicated groups of mice. (e) Densitometry analysis of ICAM-1 immunoblots; *, P < 0.001 compared with the sham groups; +, P < 0.001 compared with vehicle-treated I/R value.

Fig. 5.

IVIG protects cultured primary cortical neurons against apoptosis induced by glucose and OD. (a) Cell survival in neurons cultured with vehicle (5% sorbitol) added to the culture medium (control), culture medium containing sodium cyanide (NaCN), and culture medium containing NaCN and supplemented with 0.5% IVIG (NaCN + IVIG); neuronal survival was quantified 12 h later. Values are the mean and SEM (n = 12–18 cultures); *, P < 0.01 compared with the control value; +, P < 0.05 compared with the values for neurons exposed to NaCN. (b) Neurons were incubated for 24 h in the medium with glucose (control), in the medium lacking glucose (GD), and in the medium lacking glucose supplemented with IVIG (GD + IVIG) and albumin (GD + albumin) at 0.5%; cell viability was quantified by using Alamar blue; *, P < 0.01 compared with the control value; +, P < 0.05 compared with the values for neurons exposed to GD. (c) Immunoblot showing kinetics of increase of caspase-3 cleavage product and complement 3 (C3) in neurons exposed to combined oxygen and GD and incubated with IVIG or albumin (d). (e) Immunoblot showing the effect of C5a receptor antagonist (C5aRA) on the level of caspase-3 cleavage product in neurons subjected to GD. (f) C3 staining (red fluorescence) of cultured neurons exposed to OGD shows a signal increase at early time points relative to neurons cultured under normal conditions.