Early Erythrolysis in the Hematoma After Experimental Intracerebral Hemorrhage

Abstract

Erythrolysis occurs in the clot after intracerebral hemorrhage (ICH), and the release of hemoglobin causes brain injury, but it is unclear when such lysis occurs. The present study examined early erythrolysis in rats. ICH rats had an intracaudate injection of 100 μl autologous blood, and sham rats had a needle insertion. All rats had T2 and T2* magnetic response imaging (MRI) scanning, and brains were used for histology and CD163 (a hemoglobin scavenger receptor) and DARPP-32 (a neuronal marker) immunohistochemistry. There was marked heterogeneity within the hematoma on T2* MRI, with a hyperintense or isointense core and a hypointense periphery. Hematoxylin and eosin staining in the same animals showed significant erythrolysis in the core with the formation of erythrocyte ghosts. The degree of erythrolysis correlated with the severity of perihematomal neuronal loss. Perihematomal CD163 was increased by day 1 after ICH and may be involved in clearing hemoglobin caused by early hemolysis. Furthermore, ICH resulted in more severe erythrolysis, neuronal loss, and perihematomal CD163 upregulation in spontaneously hypertensive rats compared to Wistar-Kyoto rats. In conclusion, T2*MRI-detectable early erythrolysis occurred in the clot after ICH and activated CD163. Hypertension is associated with enhanced erythrolysis in the hematoma.

Keywords: CD163; Erythrolysis; Hematoma; Hypertension; Intracerebral hemorrhage; T2* magnetic resonance imaging.

Figure 1

Representative consecutive T2 and T2* MRIs at day 1 (A) and day 3 (B) after ICH in adult male Sprague-Dawley rats. The ratio of non-hypo-T2* volume to total T2* lesion volume was determined (C). Values are mean ± SD, n = 6.

Figure 2

Representative examples of T2 and T2* MRIs and H&E staining at the same level of the hematoma at day 1 (A) and day 3 (B) after ICH in adult male Sprague-Dawley rats. Higher magnification micrographs of the hypo-T2* (1, periphery), the border of hypo-T2* and non-hypo-T2* (2, border), the non-hypo-T2* area (3, center) of the hematoma are also shown. Scale bars are 1 mm for top panels and 10 µm for lower panels.

Figure 3

H & E staining and DARPP-32 immunoreactivity in adjacent sections at different levels of the hematoma (A). DARPP-32 staining was used to quantify neuronal loss in the ipsilateral basal ganglia and H&E staining the degree of erythrolysis and the correlation between the two parameters after ICH determined (B).

Figure 4

Immunoreactivity and quantification of CD163 positive cells in the ipsi- and contralateral caudate at day 1 (A) and day 3 (B) after ICH or a sham operation in adult male Sprague-Dawley rats. Values are mean±SD, n=6 in ICH group (per time point) and n=3 in sham-operated group (per time point). Scale bar=50µm.

Figure 5

Immunofluorescent double-labelling showed that CD163 positive cells were microglia (OX42 positive), neurons (NeuN positive) and astrocytes (GFAP positive). Scale bar=20 µm.

Figure 6

Representative consecutive T2* MRIs including the hematoma at day 1 (A) and day 3 (B) after ICH in SHR and WKY rats. The ratio of non-hypo-T2* lesion volume to total T2* lesion volume was quantified (C). Values are mean±SD, n=6. *p<0.05, #p<0.01 vs. WKY.

Figure 7

DARPP-32 immunoreactivity at day 1 (A) and day 3 (B) after ICH in SHR and WKY rats. Note the loss of DARPP-32 staining in the ipsilateral (right) caudate putamen. The loss of DARPP-32 staining (neuronal loss) was quantified as: (contralateral - ipsilateral) / contralateral DARPP-32 positive area. Values are mean±SD, n=6. Scale bar=1 mm. *p<0.05, #p<0.01 vs. WKY.

Figure 8

Immunoreactivity and quantification of CD163 positive cells in the ipsilateral caudate putamen at day 1 (A) and day 3 (B) after ICH in SHR and WKY rats. Values are mean±SD, n=6. Scale bar=50 µm. *p<0.05, #p<0.01 vs. WKY.