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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Stroke. 2021 Jun 28;52(8):2649–2660. doi: 10.1161/STROKEAHA.121.034372

The role of complement component 3 in early erythrolysis in the hematoma after experimental intracerebral hemorrhage

Ming Wang 1,2,#, Fan Xia 1,#, Shu Wan 1,2, Ya Hua 1, Richard F Keep 1, Guohua Xi 1
PMCID: PMC8316397  NIHMSID: NIHMS1711752  PMID: 34176310

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 peroxiredoxin 2 (Prx2) 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 three 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 (MRI) 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 phosphate-buffered saline and Prx2. Mice underwent both MRI 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: intracerebral hemorrhage, erythrolysis, complement C3, peroxiredoxin 2, neuroinflammation

INTRODUCTION

Erythrolysis occurs in the hematoma following intracerebral hemorrhage (ICH) and contributes to brain edema and neuronal death.1–3 Erythrolysis starts as early as the first day after ICH in animal models.1, 2 The cause of such early erythrolysis is still uncertain. However, one mechanism that can induce erythrolysis is the assembly of the lipophilic C5b-9 membrane attack complex (MAC), which forms pores on the erythrocyte membrane leading to cell lysis.4 Previous studies have shown complement activation and MAC formation occur after ICH.2, 3 It is also known that complement cascade activation and MAC formation pivots upon complement component 3 (C3) cleavage by C3-convertase.5 Our previous study found that C3-deficient mice have less neuroinflammation, microglial activation, and brain injury in the acute phase after ICH.6 The effect of C3 in erythrolysis has not been determined.

Erythrolysis in the hematoma causes the release of erythrocyte components and their degradation products into the extracellular space,3 and these can induce brain injury after ICH.1, 2 Hemoglobin (Hb), one of the most abundant erythrocyte proteins, can induce iron overload, inflammatory activation, and brain injury after ICH.7 Similarly, peroxiredoxin 2 (Prx2) is the third most abundant protein within erythrocytes8 and extracellular Prxs can elicit post-ischemic inflammation in mice,9 and Prx2 induces inflammation after experimental subarachnoid hemorrhage.10 However, the effect of Prx2 after ICH in mice has not been well studied.

This study, therefore, used C3-sufficient and deficient mice to determine the role of C3 in early erythrolysis in the hematoma, the release of Prx2 and induction of neurological deficits after ICH. It then investigated the effect of Prx2 on brain injury after ICH in mice.

METHODS

Data Availability Statement

All the data supporting this study’s results can be obtained from the corresponding author if the request is reasonable.

Animal Preparation and Intracerebral Blood Injection

Animal procedure protocols were approved by the University of Michigan Committee on Use and Care of Animals. All the animal experiments were performed under the National Institutes of Health Guide for the Care and Use of Laboratory Animals and complied with the ARRIVE reporting guidelines.

In total, 62 male C57BL/6 C3-sufficient mice and 43 C3-deficient mice (The Jackson Laboratory) aged 2~4 months were used in this study. Six C3-sufficient mice and five C3-deficient mice died during anesthesia, and another six mice were excluded from the study (five mice with small hematomas (T2* lesion<3 mm3 on day 1) and one mouse with marked intraventricular hemorrhage). Randomization was adopted by using odd/even numbers for the allocation of experimental animals. Mice anesthesia was intraperitoneally applied by using ketamine (80 mg/kg) and xylazine (5 mg/kg). A rectal probe and a feedback-controlled heating pad were used to maintain core body temperature at 37.5 °C. The right femoral artery catheterization was carried out for autologous blood collection. The mice were positioned in a stereotactic frame (Kopf Instruments), and a cranial burr hole (diameter: ~1 mm) was drilled near the right coronal suture. Then the mice received an injection of 30 μL blood into the right caudate (coordinates: 0.2 mm anterior, 3.5 mm ventral, 2.5 mm lateral to the bregma) at a rate of 3 μL/min with a 26-gauge needle.11 After injection, the needle was kept in place for 10 minutes to prevent blood reflux and then was gently removed. The bone hole was sealed with bone wax, and the scalp incision was subsequently sutured.

Experimental Groups

There were 3 parts in this study. First, C3-sufficient and deficient mice received the injection of 30 μL autologous blood into the right caudate and were euthanized on days 1, 3, 7, or 28, after completing MRI test (n=8 each group, per time point). Brains were removed for histology. Second, C3-sufficient and C3-deficient mice received 30 μL autologous blood injection. Sham mice (C3-sufficient) had only needle insertion. All the mice were euthanized on day 1. Brains were harvested for Western blotting analysis (n=4 each group). Third, C3-sufficient mice underwent an injection of 10 μg Prx2 (Novus) (dissolved in 10 μL phosphate-buffered saline, PBS) into the right caudate. Control mice received a 10 μL PBS injection. According to previous studies, the amount of Prx2 within packed erythrocytes is 5.6 mg/mL,12, 13 and this was used to determine the dose of Prx2 in the intracerebral injection. These mice were euthanized on day 1 after MRI tests. Brains were harvested for immunohistochemistry (n=6 each group).

Magnetic Resonance Imaging

Mice anesthesia was induced and maintained by a 2% isoflurane/air mixture. A forced-air heating system was applied to monitor animal body temperatures during imaging. T2- and T2*-weighted MRI was performed in a 9.4-T horizontal bore scanner (Agilent Technologies) as described previously.14, 15 Parameters for MRI were listed as follows. For T2-weighted MRI tests: field of view (FOV)=20×20 mm; slice thickness=0.5 mm, matrix (RO×PE)=256×128; repetition time (TR)=4000 ms; effective echo time (TEeffective)=60 ms. For T2*-weighted MRI tests: FOV= 20×20 mm; slice thickness=0.5 mm, matrix (RO×PE)=256×128; TR=300 ms; echo time (TE)=6 ms; flip angle (FA)=20°.

A T2* lesion was characterized as a hypointense signal area and viewed as the hematoma region. A hyperintense or isointense signal area located in the center of hematoma was defined as a non-hypo T2* lesion. The volume was measured over the total areas in all slices by multiplying the section thickness. Brain swelling was defined as ipsilateral ventricle compression and calculated on the basis of every other five sections centered on the anterior commissure layer on T2-weighted MRIs on day 3. The value is (contralateral ventricular volume-ipsilateral ventricular volume)/contralateral ventricular volume×100%. On day 28, brain tissue loss was defined as ipsilateral ventricular enlargement and calculated based on every other five sections centered on the anterior commissure layer on T2-weighted MRIs. The value is (ipsilateral ventricular volume-contralateral ventricular volume)/contralateral ventricular volume×100%. All the measurements described above were repeated three times by using ImageJ and were carried out by an investigator in a blind fashion.

Hematoxylin and Eosin Staining, Immunohistochemistry, and Immunofluorescence Staining

Mice after anesthesia underwent transcardiac perfusion with 4% paraformaldehyde (PFA). Brains were then soaked in 4% PFA, dehydrated with 30% glucose, and sectioned coronally (18-μm-thick slices). Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence staining were conducted in accordance with previously described protocols.16 For immunostaining, the primary antibodies were rabbit anti-DARPP-32 (1:400, Cell Signaling Technology), rabbit anti-heme oxygenase-1 (HO-1) (1:800, Enzo), rabbit anti-ferritin (1:500, Sigma), goat anti-macrophage scavenger receptor-1 (MSR-1) (1:100, R&D Systems), rat anti-CD4 (1:200, BioLegend), and goat anti-albumin (1:1000, Bethyl Laboratories Inc).

HO-1, ferritin, MSR-1, and CD4-positive cells were counted separately in four different regions, including the hematoma, perihematomal tissue, ipsilateral basal ganglia (BG) tissue (50 μm from perihematomal tissue), and contralateral BG tissue. Three different areas were selected in each region from every other two sections in high-power images. The counting results were calculated by an average of the number of positive cells in the three selected regions and were demonstrated as cells per square millimeter. Bilateral DARPP-32-positive areas were outlined, and ipsilateral loss was measured as the neuronal loss. The value was (contralateral hemisphere-ipsilateral hemisphere)/contralateral hemisphere×100%. The albumin-positive areas across the whole brain sections were outlined, and the albumin area ratio was measured by albumin-positive area/whole brain section area×100%. All the measurements were repeated three times with ImageJ by an investigator in a blind fashion.

Fluoro-Jade C Staining

Fluoro-Jade C staining was used to evaluate neuronal degeneration as previously described.17 Brain sections were dried on a warmer at 50°C for 1 hour. Then 0.06% potassium permanganate was used for incubation for 15 minutes. After a rinse technique, brain sections were incubated with 0.001% Fluoro-Jade C in 1% acetic acid for 10 minutes on a shaker in the dark. Then sections were rinsed, air-dried, and subsequently cleared in xylene and were mounted.

Western Blot Analysis

Western blot analysis was performed as previously described.2, 11, 16 Mice after anesthesia underwent transcardiac perfusion with ice-cold 0.1 mol/L PBS. Brains were then harvested and cut parallelly to the midline through needle insertion point on the brain surface. The hematoma was then removed, and the tissue surface surround the hematoma was rinsed with ice-cold 0.1 mol/L PBS and at least 1 mm thick peri-hematoma tissues which were in direct contact with the hematoma were carefully scraped away. Then the ipsilateral BG with the remaining perihematomal tissue was sampled. To sample the contents of erythrocytes, autologous blood was washed in saline for 3 times, and packed erythrocytes were obtained by centrifugation. Erythrocyte pellets were disrupted by ultrasound and then sampled. The concentration of protein solution was measured by the Bio-Rad protein assay kit (Hercules). Then 50 μg protein samples were suspended and loaded on a 12.5% SDS-PAGE gel. After transferred protein onto a Hybond-C PURE Nitrocellulose membrane (Amersham), 5% non-fat milk (LabScientific Inc) was used for 1-hour blockade. Then the membrane was incubated with primary and secondary antibodies. The primary antibodies were rabbit anti-peroxiredoxin 2 (1:5000, Novus), rabbit anti-hemoglobin subunit α (1:2500, Abcam), and mouse anti-β-actin (1:40000, Sigma). The second antibodies were goat anti-rabbit IgG (1:7500, Bio-Rad) and goat anti-mouse IgG (1:7500, Bio-Rad). ECL chemiluminescence system (Amersham) and Kodak X-OMAT film (Sigma) were used for final visualization. The relative densities of bands were analyzed with ImageJ.

Behavioral Tests

Corner turn and forelimb use asymmetry tests were carried out for mice behavioral evaluation as previously described.18 Animals were evaluated before the surgery and on 1, 3, 7, and 28 days after ICH, and on 1 day after Prx2 injection. All the tests were conducted by an investigator blindly.

Statistical analysis

All the numerical data are analyzed by Student’s t-test or one-way ANOVA with Tukey post hoc test and presented as means±SD. Statistical significance was set at P < 0.05. Data from our prior study6 indicated that n=7 would have 80% power to detect a 40% reduction in both brain edema and forelimb asymmetry score with a type I error rate (alpha) of 5%. An n=8 was chosen in case of any procedural dropout.

RESULTS

C3-deficient mice had less early erythrolysis in the hematoma

Isointense or hyperintense areas (non-hypo-T2* lesion) in the hematoma were observed on day 3 after ICH in both C3-sufficient and deficient mice but were not evident on day 1 after ICH. Our previous studies have confirmed that the non-hypo-T2* lesion correlates with erythrolysis.1–3 The ratio of non-hypo T2* lesion to total T2* lesion volume was calculated. The results showed that C3-deficient mice had significantly less erythrolysis (9.1±4.2 vs. 16.8±4.8% in C3-sufficient mice, P < 0.01; Figure 1A).

Figure 1.

Figure 1.

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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.

Brain swelling and neuronal degeneration were less in C3-deficient mice on day 3 after ICH

Bilateral ventricular volume was measured on T2-weighted MRI. The degree of ipsilateral ventricular compression was used as a measure of brain swelling. Ipsilateral ventricular compression was significantly less in C3-deficient mice on day 3 after ICH (46.1±15.2 vs. 58.4±11.6% in C3-sufficient mice, P < 0.01; Figure 1B). DARRP-32 immunohistochemistry stains striatal neurons. The loss of DARRP-32 positive staining was less in C3-deficient mice (16.2±8.8 vs. 26.6±10.1% in C3-sufficient mice, P < 0.05; Figure 1C) on day 3 after ICH. Fluoro-Jade C staining was also applied to identify neuronal degeneration. The Fluoro-Jade C positive cells in ipsilateral basal ganglia were fewer in C3-deficient mice (270±120 vs. 552±189 cells/mm2 in C3-sufficient mice, P < 0.01; Figure 1D) at day 3 after ICH.

Long-term brain atrophy and neurological deficits after ICH were less severe in C3-deficient mice

The ipsilateral ventricular enlargement was measured on T2-weighted MRI on day 28 following ICH to assess brain atrophy. The ipsilateral ventricle changed less in C3-deficient mice (6.2±7.7 vs. 41.3±9.5% in C3-sufficient mice, P < 0.01; Figure 2A). The loss of DARRP-32 positive area was also less in C3-deficient mice (19.8±7.1 vs. 27.8±7.8% in C3-sufficient group, P < 0.05; Figure 2B) on day 28. In addition, C3-deficient mice had better forelimb use asymmetry scores on days 3, 7, and 28 (Figure 2C), and better corner turn scores on day 3, 7, and 28 (Figure 2D).

Figure 2.

Figure 2.

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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.

C3-deficient mice had fewer HO-1- and ferritin-positive cells after ICH

Erythrolysis causes erythrocyte proteins to be released into the extracellular space. Hb released after erythrolysis is taken up by macrophages and then degraded by heme oxygenases (HOs), further producing iron, biliverdin, and carbon monoxide.3 Ferritin is induced to bind intracellular iron.19 Immunohistochemistry demonstrated that the HO-1-positive cells were significantly fewer in the hematoma, perihematomal tissue, and ipsilateral BG tissue in C3-deficient mice on day 3 after ICH (hematoma: 251±102 vs. 607±18 cells/mm2 in C3-sufficient mice, P < 0.01; perihematomal tissue: 637±143 vs. 1025±182 cells/mm2 in C3-sufficient mice, P < 0.01; ipsilateral BG: 376±125 vs. 557±82 in C3-sufficient mice, P < 0.01; Figure 3A).

Figure 3.

Figure 3.

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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.

Ferritin-positive cells were fewer in perihematomal tissue in C3-deficient mice on day 7 after ICH (718±335 vs. 1075±1203 cells/mm2 in C3-sufficient mice, P < 0.05). However, there were no significant differences in hematoma or ipsilateral BG tissue (P > 0.05) (Figure 3B).

C3-deficient mice had less Prx2 released from erythrocytes and fewer MSR-1- and CD4-positive cells infiltration after ICH

Prx2 is the third abundant protein within erythrocytes and it is released following erythrolysis.20 In the current study, Prx2 and hemoglobin levels in the erythrocytes were first determined by Western blot analysis (Prx2: 12341 ± 1710 vs. 12381 ± 2945 pixels in C3-sufficient mice, n=4, P > 0.05; Hemoglobin subunit α: 18524±974 vs. 17928±2521 pixels in C3-sufficient mice, n=4, P > 0.05; Prx2 to hemoglobin subunit α ratio: 0.67±0.08 vs. 0.69±0.11 in C3-sufficient mice, P > 0.05). There were no significant differences in erythrocyte Prx2 levels between C3-sufficient and deficient mice. Then Prx2 levels in a perihematomal and ipsilateral BG combined sample were determined on day 1 after ICH. There was less Prx2 protein release in C3-deficient mice (Prx2/β-actin: 0.43 ± 0.12 vs. 1.18 ±0.09 in C3-sufficient mice, P < 0.01; Figure 4A). A prior study has identified MSR1-positive cells as being responsible for the clearance of damage-associated molecular patterns (DAMPs).21 As extracellular Prx2 is a potent pro-inflammatory factor,22 alterations in MSR-1 positive cells may closely correlate with the level of extracellular Prx2. In concert with less Prx2 release from erythrocytes after ICH, there were MSR-1-positive cells in hematoma, perihematomal tissue, and ipsilateral BG tissue in C3-deficient mice on day 3 after ICH (hematoma: 122±74 vs. 337±95 cells/mm2 in C3-sufficient mice, P < 0.01; perihematomal tissue: 333±116 vs. 637±148 cells/mm2 in C3-sufficient mice, P < 0.01; ipsilateral BG: 72±85 vs. 322±122 in C3-sufficient mice, P < 0.01; Figure 4B). Some, but not all, MSR-1 positive cells were also HO-1 positive as assessed by immunofluorescence co-localization. (Supplementary Figure I). CD4-positive T lymphocytes are the predominant brain leukocyte population after ICH in mice.23 There is a strong relationship between Prx2 release following erythrolysis and leucocyte recruitment after hemorrhagic stroke.22, 24 In the present study, the number of CD4-positive cells was significantly decreased in hematoma, perihematomal tissue and ipsilateral BG tissue in C3-deficient mice on day 3 after ICH (hematoma: 62±40 vs. 180±53 cells/mm2 in C3-sufficient mice, P < 0.01; perihematomal tissue: 63±55 vs. 357±166 cells/mm2 in C3-sufficient mice, P < 0.01; ipsilateral BG: 16±12 vs. 98±45 in C3-sufficient mice, P < 0.01; Figure 4C).

Figure 4.

Figure 4.

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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.

Prx2 induced blood-brain barrier disruption, brain edema, and neuronal injury, recruited MSR-1- and CD4-positive cells, and caused neurological impairment

In order to determine whether Prx2 can induce brain damage and recruit MSR-1and CD4-positive cells, as found following erythrolysis after ICH, Prx2 was injected into the mice right BG. T2-weighted MRI on day 1 after injection found that Prx2 induces brain edema (22.5±9.7% vs. 6.0±4.6% in control mice, P < 0.01; Figure 5A). Besides that, Prx2-injected mice had more FJC-positive cells in the ipsilateral basal ganglia (376±115 cells/mm2 vs. 176±51 cells/mm2 in control mice, P < 0.01; Figure 5B). Albumin immunohistochemistry was used to assess blood-brain barrier leakage after injection. The albumin area ratio was much higher in Prx2-injected mice (50.0±11.2% vs. 5.6±2.7% in control mice, P < 0.01; Figure 5C). Blood-brain barrier disruption and neuronal degeneration may recruit macrophages and induce leukocyte infiltration.24 The MSR-1-positive cells were evidently recruited after the injection of Prx2 (354±160 vs. 59±37 cells/mm2 in control mice, P < 0.01; Figure 6A). Besides that, there was also a significant increase in the CD4-positive cells around the Prx2 injection site (134±78 vs. 29±19 cells/mm2 in control mice, P < 0.01; Figure 6B). Behavioral tests demonstrated that the Prx2-injected mice had more severe neurological deficits (forelimb use asymmetry score: 30.7±13.0 vs.9.9±9.5 in control mice, P < 0.01; corner turn tests: 73.3±10.3 vs.53.3±12.1 in control mice, P < 0.01; Figure 6C and 6D).

Figure 5.

Figure 5.

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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.

Figure 6.

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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.

DISCUSSION

The major findings of this study are: (1) Erythrolysis, ICH-induced brain injury, and neurological deficits after ICH were less severe in C3-deficient mice; (2) Less Prx2 was released from the hematoma early following ICH in C3-deficient mice; (3) Prx2 induced blood-brain barrier disruption, brain edema, neuronal injury, and neurological deficits in mice; (4) Prx2 recruited MSR-1- and CD4-positive cells to brain.

Our previous studies have uncovered that early erythrolysis occurs in both piglet and rat ICH models.1, 2 We found a transformation of erythrocytes in the center of the hematoma from discocyte to spherocyte starting on day 3 after ICH in a piglet model,2 but as early as day 1 after ICH in rat models.1 T2* sequence MRI, a non-invasive examination, can identify erythrolysis in the hematoma. Erythrolysis presents as a hyperintense or isointense signal (non-hypo T2* lesion) on T2* MRI,1, 25, 26 and thus T2* MRI was used to detect erythrolysis in the mouse ICH models in the current study. Non-hypo T2* lesions in the hematoma were observed on day 3 but were not marked on day 1 after ICH. This phenomenon suggests that the occurrence of erythrolysis in after ICH in mice is similar to piglets, but later than in rats.

The mechanisms of erythrolysis in the hematoma after ICH remain incompletely elucidated. Complement activation and MAC formation on the erythrocyte membrane play a crucial part in systemic erythrolysis.27, 28 Our prior work has also identified complement activation after ICH and erythrolysis in hematoma accompanied by MAC accumulation.2, 4 Recent studies have also indicated that complement not only plays an essential part in the pathogenesis of secondary injury in the acute and subacute phases of stroke, but also appears to have significant implications regarding neurological recovery in chronic the phase.29, 30 It is known that all complement pathways converge at complement protein C3. Cleavage of C3 is the key step in MAC formation.5 Therefore, we hypothesized that C3 might significantly affect erythrolysis in the hematoma following ICH. Indeed, the present study demonstrated that C3-deficient mice had significantly less erythrolysis compared with C3-sufficient mice. Subsequently, neuroinflammation and microglia activation in the acute phase after ICH were reduced in C3-deficient mice.6 Moreover, it also demonstrated that C3-deficient mice not only had less brain edema and neuronal degeneration in the acute phase after ICH. In addition, they had less severe long-term brain atrophy and better functional outcomes.

Erythrolysis within the hematoma causes the release of cytoplasmic proteins into the extracellular space. Such proteins, and their degradation products, can induce brain injury. Hb and Prx2 are the first and third most abundant erythrocyte proteins.8 After erythrolysis causes the release of Hb into extracellular space, it is degraded by HO-1 in the macrophages/microglia or HO-2 in the neurons into carbon monoxide, biliverdin, and Fe2+.3 In macrophages/microglia, iron is bound to ferritin limiting its toxicity. However, a relative lack of ferritin in neurons makes them susceptible to iron overload and injury.7, 31–34 In the current study, we identified that C3-deficient mice had less erythrolysis. Additionally, the expression of HO-1 and ferritin was also less in C3-deficient mice as would be expected if there is less release of Hb from the hematoma.

This study also found less Prx2 release into the tissue around the hematoma in C3-deficient mice, again consistent with there being less erythrolysis. Other than Hb, the effects of erythrocyte proteins on brain injury after ICH have not been as well studied in mouse models. Prxs are a family of antioxidant enzymes essential for maintaining cell signal regulation and cell survival.35 Therefore, Prx2 could have some beneficial function. However, they can cause post-ischemic inflammation when released into extracellular space. Extracellular Prxs function as DAMPs, inducing IL-23 expression via TLR2 and TLR4,21 and a similar effect of Prx2 was also found in experimental subarachnoid hemorrhage.10 The present study found that Prx2 could induce brain edema, BBB disruption, and neuronal degeneration and result in severe neurological deficits in mice.

Prx2 also recruits MSR-1- and CD4-positive cells into the lesion. Prior research indicates that Prxs are internalized through the class A scavenger receptor MSR-1 in experimental ischemic stroke in mice.21 Our previous investigation found that MSR-1-positive cells infiltrate into the hematoma along the white matter fibers and might function as a key phagocytic factor on mononuclear phagocytes after ICH in piglets.36 However, the function of MSR-1-positive cells in mouse experimental ICH is yet to be determined. Here, our findings indicate that Prx2 is capable of recruiting MSR-1-positive cells following ICH, although other substances released post-erythrolysis might also upregulate MSR-1 expression. This warrants further investigations. CD4-positive cells and granulocytes are predominant leukocyte populations in early ICH.23 Our previous studies demonstrated that the main granulocytic subpopulation, neutrophils, infiltrated into the lesion site both in intraparenchymal and intraventricular Prx2 injection models.22, 24 The current study found that CD4-positive cells were another brain-invading leukocyte population that had a close correlation with the release of Prx2 following ICH in mice. Taken together, these results indicate that targeting post-ICH release of Prx2 from erythrocytes might serve as an effective therapeutic approach for alleviating brain injury.

While the evidence presented in this study implicates C3-mediated RBC lysis in ICH-induced brain injury, it should be noted that there are likely other roles of C3 in injury, such as other potential proinflammatory mechanisms. The relative importance of these different mechanisms still needs to be investigated (e.g. by using other methods to block RBC lysis or examining the effects of C3 on the brain injury induced by RBC lysate).

The current study has several limitations that should be addressed. 1) It examined the effect of C3 in erythrolysis and the release of Prx2 after ICH in male mice. More studies are needed to investigate the effect in female mice; 2) Only one dose of Prx2 was tested here and this should be expanded to different doses as this might reflect effects at different hematoma sizes; 3) When quantifying HO-1 and ferritin positive cells, ramified and amoeboid cells were not distinguished; 4) The current study demonstrated that Prx2 could induce brain injury in the mice, but the exact mechanism of Prx2-induced brain injury after ICH, such as T cell-mediated immune responses, as well as the role of Prx2 degradation products, needs to be investigated in further studies.

In summary, this study demonstrates that early erythrolysis within the hematoma occurs by day 3 after ICH mice and that it is less in C3-deficient mice. C3-deficient mice have less Prx2 release from erythrocytes and reduced brain injury after ICH. In addition, Prx2 release following erythrolysis can cause brain damage in mice and recruit MSR-1- and CD4-positive cells. Targeting erythrolysis and the effect of extracellular Prx2 are potential therapeutic strategies in ICH.

Supplementary Material

Supplemental Publication Material

Acknowledgments

We appreciate BioRender.com for help with the graphic abstract illustration.

Sources of Funding

YH, RFK, and GX were supported by grants NS-091545, NS-090925, NS-096917, NS106746 and NS116786 from the National Institutes of Health (NIH).

Footnotes

Disclosures

All authors declare no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Publication Material
NIHMS1711752-supplement-Supplemental_Publication_Material.pdf (12MB, pdf)

Data Availability Statement

All the data supporting this study’s results can be obtained from the corresponding author if the request is reasonable.

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