Enhancement of Hematoma Clearance With CD47 Blocking Antibody in Experimental Intracerebral Hemorrhage

Abstract

Background and Purpose- Our previous studies found that erythrocyte CD47 has a role in regulating hematoma resolution following experimental intracerebral hemorrhage (ICH). The current study examined whether or not a CD47 blocking antibody enhances hematoma clearance in a mouse ICH. Methods- ICH was induced by intracaudate injection of autologous blood in adult C57BL/6 mice. Mice had an ICH or ICH with CD47 blocking antibody or IgG coinjection. In subgroups of CD47 blocking antibody-treated mice, clodronate (to deplete microglia/macrophages) or control liposomes were coinjected. The effects of CD47 blocking antibody on ICH-induced brain injury were also tested in both males and females. Mice had magnetic resonance imaging to examine clot volume, iron deposition, brain swelling, and brain tissue loss. Behavioral tests were performed in all mice, and brains were harvested for brain immunohistochemistry. Results- In male mice, CD47 blocking antibody speeded up hematoma/iron clearance by macrophages/microglia and reduced ICH-induced brain swelling, neuronal loss, and neurological deficits. In contrast, clodronate liposome-induced microglia/macrophage depletion caused more severe brain swelling, neuronal loss, and functional deficits. In addition, similar injury severity in males and females was found in IgG control group and CD47 blocking antibody was also effective in females. Conclusions- Blocking CD47 in the hematoma speeded hematoma clearance and reduced brain injury after ICH suggesting it could be a treatment for ICH patients with surgical clot removal.

Keywords: cerebral hemorrhage; erythrocytes; macrophages; mice; microglia.

Figure 1.

(A) Time course of T2* lesion after ICH (30-μl blood injection) in male mice. Values are means ± SD; n=6; # P<0.01 vs. day 1 by one way ANOVA. (B) Time course of T2* lesion after co-injection of blood and anti-CD47 antibody or IgG. Residual hematoma/brain iron are presented as the ratio of T2* lesion volumes at day 7 or day 28 to day 1. Values are means ± SD; n=12–22; two-way ANOVA; # P<0.01 vs. day 1, *P<0.01 vs. ICH+IgG group.

Figure 2.

(A) T2 MRI assessment of brain swelling at day 1 after saline, ICH+anti-CD47 antibody or ICH+IgG. Values are means ± SD; n=10 for saline, n=22 for both ICH+anti-CD47 and ICH+IgG; # P<0.01 vs other groups by one way ANOVA. (B) Representative DARPP-32 immunoreactivity at day 28 after saline, anti-CD47 antibody or IgG mixed blood injection. Values are means±SD; n=6 for each group; #P<0.01 vs. other groups by one way ANOVA. (C) Corner turn and forelimb use asymmetry scores before and after ICH or saline injection. Values are means±SD; ICH groups: n=22 for pre-test, days 1 and 7, n=12 for day 28; saline control n=6, all time points; # P<0.01 vs other groups by two-way ANOVA.

Figure 3.

(A) Positive cells of MOMA-2 in different locations at day 7 after co-injection of blood with anti-CD47 antibody or IgG. The high magnification images correspond to the hematoma, peri-hematoma edge and the ipsilateral basal ganglia. Upper scale bar =200 μm, lower scale bar = 20μm. Values are means±SD; n=6 for each group; # P<0.01 vs ICH+IgG group by student t test. (B) HO-1 positive cells in different locations at day 7 after co-injection of blood with anti-CD47 antibody or IgG. The high magnification images correspond to the hematoma, peri-hematoma edge and ipsi-basal ganglia. Upper scale bar =200 μm, lower scale bar = 20μm. Values are means±SD; n=6 for each group; # P<0.01 vs IgG treated group by student t test.

Figure 4.

(A) Effect of clodronate or control liposomes on MOMA-2 immunoreactivity after ICH in anti-CD47 antibody treated mice. Two examples of low magnification images of MOMA-2 positive staining are shown for each treatment. The high magnification images correspond to the hematoma, peri-hematoma edge and ipsilateral basal ganglia. Upper scale bar =200 μm, lower scale bar = 20μm. Quantification of the number of MOMA-2 positive cells in different brain regions. Values are means±SD; n=7 for each group; # P<0.01 vs control liposome treated group by student t test. (B) HO-1 positive cells of in difference locations at day 7 after co-injection of clodronate or control liposome with anti-CD47 antibody mixed with blood. The high magnification images correspond to the hematoma, peri-hematoma edge and ipsilateral basal ganglia. Upper scale bar =200 μm, lower scale bar = 20μm. Values are means±SD; n=7 for each group; # P<0.01 vs control liposome treated group by student t test.

Figure 5.

(A) Effect of clodronate or control liposomes on hematoma resolution after ICH in anti-CD47 antibody treated mice. Representative T2* weighted MRI after co-injection of clodronate or control liposome with anti-CD47 antibody and blood. Bar graphs show residual hematoma rate at day 7. Values are means±SD; n=9 for each group; # P<0.01 vs anti-CD47+Control group by student t test. (B) Representative T2 weighted MRI showing brain swelling at day 1 after ICH in the two groups and quantification. Values are means±SD; n=9 for each group; # P<0.01 vs. anti-CD47+Control group by student t test. (C) Representative DARPP-32 immunoreactivity at day 7 in the two groups and quantification. Values are means±SD; n=9 for each group; # P<0.01 vs. anti-CD47+Control group by student t test. (D) Corner turn score and forelimb use asymmetry before and after ICH in the two groups. Values are means±SD; n=9 for each group; # P<0.01 vs. anti-CD47+Control group by two-way ANOVA.

Figure 6.

(A) Representative T2* weighted MRI after blood injection in female mice treated with IgG or anti-CD47 antibody. Residual hematoma volumes are presented at day 7 (ratio to day 1). Values are means±SD; n=6 for each group; # P<0.01 vs IgG treated group by student t test. (B) T2 MRI assessment of brain swelling at day 1 after ICH in female mice. Values are means±SD; n=6 for each group; * P<0.05 vs IgG treated group by student t test. (C) DARPP-32 immunoreactivity at day 7 after injection in female mice. Values are means±SD; * P<0.05 vs IgG treated group by student t test. (D) Corner turn and forelimb use asymmetry tests before and after injection in female mice. Values are means±SD; n=6 for each group; # P<0.01 vs. IgG treated group by two-way ANOVA.