Hematoma Changes During Clot Resolution After Experimental Intracerebral Hemorrhage

Abstract

Background and purpose: Hematoma clearance occurs in the days after intracerebral hemorrhage (ICH) and has not been well studied. In the current study, we examined changes in the hematoma in a piglet ICH model. The effect of deferoxamine on hematoma was also examined.

Methods: The ICH model was induced by an injection of autologous blood into the right frontal lobe of piglets. First, a natural time course of hematoma changes ≤7 days was determined. Second, the effect of deferoxamine on hematoma changes was examined. Hemoglobin and membrane attack complex levels in the hematoma were examined by enzyme-linked immunosorbent assay. Immunohistochemistry and Western blotting were used to examine CD47 (a regulator of erythrophagocytosis), CD163 (a hemoglobin scavenger receptor), and heme oxygenase-1 (a heme degradation enzyme) in the clot.

Results: After ICH, there was a reduction in red blood cell diameter within the clot with time. This was accompanied by membrane attack complex accumulation and decreased hemoglobin levels. Erythrophagocytosis occurred in the hematoma, and this was associated with reduced clot CD47 levels. Activated macrophages/microglia were CD163 and hemeoxygenase-1 positive, and these accumulated in the clot with time. Deferoxamine treatment attenuated the process of hematoma resolution by reducing member attack complex formation and inhibiting CD47 loss in the clot.

Conclusions: These results indicate that membrane attack complex and erythrophagocytosis contribute to hematoma clearance after ICH, which can be altered by deferoxamine treatment.

Keywords: cerebral hemorrhage; deferoxamine; hemolysis; membrane attack complex; swine.

Conflict of interest statement

Potential Conflicts of Interest: We declare that we have no conflict of interest.

© 2016 American Heart Association, Inc.

Figures

Figure 1

(A) Coronal sections of perfused piglet brain showing hematomas. (B) Time course of clot changes in hematoma edge and center. Scale bar = 10 μm, scale bar = 2.5 μm in the insets. (C) Diameter of RBC in hematoma edge and center. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour.

Figure 2

(A) Coronal section of an in situ frozen piglet brain after ICH. The square represents the sample area. (B) Time course of hemoglobin content in hematoma. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour. (C) Time course of MAC content in the hematoma. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour.

Figure 3

(A) Erythrophagocytosis (arrows) at day-3 and day-7 in the hematoma and hemosiderin deposition (arrowhead) at day-7. Scale bar = 10 μm. (B) Time course of CD47 levels in the hematoma. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour.

Figure 4

(A) Time course of CD163 immunoreactivity and number of positive cells in the hematoma. Upper scale bar = 50μm, lower scale bar = 10μm. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour. (B) Time course of HO-1 immunoreactivity and protein levels. Upper scale bar = 50 μm, lower scale bar = 10 μm. Values are mean ± SD, *p <0.05, #p <0.01 vs. 4-hour.

Figure 5

(A) Hemoglobin, (B) MAC and (C) CD47 protein levels in the hematoma in vehicle- and DFX-treated groups at 3 days after ICH. Values are mean ± SD, *p <0.05 vs. vehicle treated group.

Figure 6

(A) CD163 immunoreactivity and numbers of positive cells in the hematoma in vehicle- and DFX-treated groups at 3 days after ICH. Upper scale bar = 50 μm, lower scale bar = 10 μm. Values are mean ± SD, *p <0.05. (B) HO-1 immunoreactivity and proteins levels in the hematoma in vehicle- and DFX-treated groups at 3 days after ICH. Upper scale bar = 50 μm, lower scale bar =10 μm. Values are mean ± SD, *p <0.05.