Role of Complement Component 3 in Early Erythrolysis in the Hematoma After Experimental Intracerebral Hemorrhage

Abstract

Background and purpose: Early erythrolysis occurs within the hematoma following intracerebral hemorrhage (ICH), and the release of erythrocyte cytoplasmic proteins such as hemoglobin and Prx2 (peroxiredoxin 2) can cause brain injury. Complement activation can induce erythrolysis. This study determined the function of complement component 3 (C3) in erythrolysis in hematoma and brain injury after ICH in mice.

Methods: This study has 3 parts. First, ICH was induced in adult male C3-sufficient and deficient mice and animals were euthanized on days 1, 3, 7, and 28 for immunohistochemistry after magnetic resonance imaging and behavioral testing. Second, C3-sufficient and deficient mice with ICH were euthanized on day 1 for Western blot analysis. Third, C3-sufficient mice received injections of PBS and Prx2. Mice underwent both magnetic resonance imaging and behavioral tests on day 1 and were then euthanized. Brains were harvested for immunohistochemistry and Fluoro-Jade C staining.

Results: Erythrolysis occurred in the hematoma in C3-sufficient and deficient mice on day 3 following ICH. C3-deficient mice had less erythrolysis, brain swelling, and neuronal degeneration in the acute phase and less brain atrophy in the chronic phase. There were fewer neurological deficits on days 3, 7, and 28 in C3-deficient mice. C3-deficient mice also had less extracellular Prx2 release. Moreover, Prx2 induced brain edema and brain injury and recruited macrophage scavenger receptor-1- and CD4-positive cells following ICH in mice.

Conclusions: C3-deficient mice had less severe erythrolysis and brain injury following ICH compared with C3-sufficient mice. Prx2 released after erythrolysis can cause brain damage and neuroinflammation in mice.

Keywords: atrophy; brain; hematoma; hemoglobin; mice.

Figure 1.

Erythrolysis within hematoma, brain edema, and neuronal loss/degeneration on day 3 after ICH in C3-sufficient and deficient mice. (A) Representative consecutive T2* MRIs on day 3 after ICH in C3-sufficient and deficient mice. The ratio of non-hypo T2* lesion to total T2* lesion volume was calculated. Yellow dashed lines represented non-hypo areas. Values are presented as means±SD. n=24. #P<0.01 vs. C3-sufficient mice. (B) Representative consecutive T2 MRIs on day 3 after ICH in C-sufficient and deficient mice. Brain swelling was evaluated by ipsilateral ventricular compression. Values are presented as means±SD. n=24. #P<0.01 vs. C3-sufficient mice. (C) DARPP-32 immunohistochemistry staining on day 3 after ICH was used to evaluate neuronal loss. Values are presented as means±SD. n=8. Scale bar=1 mm. *P<0.05 vs. C3-sufficient mice. (D) Fluoro-Jade C staining on day 3 after ICH was used to evaluate neuronal degeneration. Values are presented as means±SD. n=8. * indicates hematoma area. Scale bars are 100 μm in low magnification and 20 μm in high magnification. #P<0.01 vs. C3-sufficient mice.

Figure 2.

Brain atrophy and neuronal loss on day 28 after ICH as well as functional outcome after ICH in C3-sufficient and deficient mice. (A) Representative T2 MRIs on day 28 after ICH in C3-sufficient and deficient mice. Ipsilateral ventricle enlargement was quantified. Values are presented as means±SD. n=8. #P<0.01 vs. C3-sufficient mice. (B) DARPP-32 immunohistochemistry staining on day 28 after ICH for neuronal loss evaluation. Values are presented as means±SD. n=8. Scale bar=1 mm. *P<0.05 vs. C3-sufficient mice. Scores of (C) forelimb use asymmetry and (D) corner turn tests pre-ICH (n=36 each group) and on day 1 (n=32 each group), 3 (n=24 each group), 7 (n=16 each group), and 28 (n=8 each group) after ICH in C3-sufficient and deficient mice. Values are presented as means±SD. #P<0.01, *P<0.05 vs. C3-sufficient mice.

Figure 3.

The expression of HO-1 and ferritin in C3-sufficient and deficient mice. (A) Diagram showing cell counting locations. (B) HO-1 immunoreactivity in hematoma, perihematomal tissue, ipsilateral basal ganglia, and contralateral basal ganglia on day 3 after ICH. Values are presented as means±SD. n=8. Scale bar=20 μm. #P<0.01 vs. C3-sufficient mice. (C) Ferritin immunoreactivity in hematoma, perihematomal tissue, ipsilateral basal ganglia, and contralateral basal ganglia on day 7 after ICH. Values are presented as means±SD. n=8. Scale bar=20 μm. *P<0.05 vs. C3-sufficient mice.

Figure 4.

Prx2 release following erythrolysis and the expression of MSR-1 and CD4 after ICH in C3-sufficient and deficient mice. (A) Prx2 levels in the perihematomal tissue and ipsilateral basal ganglia (combined sample) on day 1 after ICH in C3-sufficient and deficient mice or a sham operation (in C3-sufficient mice). Values are presented as means±SD. n=4. #P<0.01 vs. other groups. (B) MSR-1 immunoreactivity in hematoma, perihematomal tissue, ipsilateral basal ganglia, and contralateral basal ganglia on day 3 after ICH in C3-sufficient and deficient mice. Values are presented as means±SD. n=8. Scale bar=20 μm. #P<0.01 vs. C3-sufficient mice. (C) CD4 immunoreactivity in hematoma, perihematomal tissue, ipsilateral basal ganglia, and contralateral basal ganglia on day 3 after ICH in C3-sufficient and deficient mice. Values are presented as means±SD. n=8. Scale bar=20 μm. #P<0.01 vs. C3-sufficient mice.

Figure 5.

Brain edema, neuronal degeneration, and blood-brain barrier disruption after Prx2 injection in mice. (A) Representative T2 MRIs on day 1 after 10 μg Prx2 injection (in 10 μL PBS). Control mice received an injection of 10 μL PBS. Brain swelling was evaluated by ipsilateral ventricular compression. Values are presented as means±SD. n=6. #P<0.01 vs. control group. (B) Fluoro-Jade C staining in the ipsilateral basal ganglia on day 1 after Prx2 injection. Control mice received a PBS injection. Values are presented as means±SD. n=6. Scale bar was 100 μm in low magnification and 20 μm in high magnification. #P<0.01 vs. control group. (C) Albumin immunoreactivity on day 1 after Prx2 injection. Control mice received a PBS injection. Values are presented as means±SD. n=6. Scale bar=1 mm. #P<0.01 vs. control group.

Figure 6.

Prx2 injection induces MSR-1- and CD4-positive cells infiltration into brain and neurological deficits in mice. (A) MSR-1 and (B) CD4 immunoreactivity in the ipsilateral basal ganglia on day 1 after Prx2 injection. Control mice received a PBS injection. Values are presented as means±SD. n=6. Scale bar was 200 μm in low magnification and 20 μm in high magnification. #P<0.01 vs. control group. Scores of (C) forelimb use asymmetry and (D) corner turn tests pre-injection and on day 1 after Prx2 injection. Control animals received PBS injection. Values are presented as means±SD. n=6. #P<0.01 vs. control group.