The absence of the CD163 receptor has distinct temporal influences on intracerebral hemorrhage outcomes

Abstract

Hemoglobin (Hb) toxicity precipitates secondary brain damage following intracerebral hemorrhage (ICH). CD163 is an anti-inflammatory Hb scavenger receptor and CD163-positive macrophages/microglia locally accumulate post-bleed, yet no studies have investigated the role of CD163 after ICH. ICH was induced in wildtype and CD163^-/- mice and various anatomical and functional outcomes were assessed. At 3 d, CD163^-/- mice have 43.4 ± 5.0% (p = 0.0002) and 34.8 ± 3.4% (p = 0.0003) less hematoma volume and tissue injury, respectively. Whereas, at 10 d, CD163^-/- mice have 49.2 ± 15.0% larger lesions (p = 0.0385). An inflection point was identified, where CD163^-/- mice perform better on neurobehavioral testing and have less mortality before 4 d, but increased mortality and worse function after 4 d (p = 0.0389). At 3 d, CD163^-/- mice have less Hb, iron, and blood-brain barrier dysfunction, increased astrogliosis and neovascularization, and no change in heme oxygenase 1 (HO1) expression. At 10 d, CD163^-/- mice have increased iron and VEGF immunoreactivity, but no significant change in HO1 or astrogliosis. These novel findings reveal that CD163 deficiency has distinct temporal influences following ICH, with early beneficial properties but delayed injurious effects. While it is unclear why CD163 deficiency is initially beneficial, the late injurious effects are consistent with the key anti-inflammatory role of CD163 in the recovery phase of tissue damage.

Keywords: Gliosis; heme oxygenase; iron; oxidative stress; stroke.

Figure 1.

CD163 deficiency temporally influences ICH-induced brain damage. Representative Cresyl violet stained brain sections are shown for WT and CD163^−/− mice at (a) 3 d and (g) 10 d after collagenase-induced ICH. Within genotype and study endpoint, images are from the same animal, where left-to-right corresponds to anterior-to-posterior sections. Representative high magnification images are shown for (b) blood content at 3 d and (h) hematoidin content at 10 d for WT and CD163^−/− mice. At 3 d, CD163^−/− mice have significantly less (c) overall ICH-induced brain damage, (d) residual blood volume, and (e) tissue injury. (f) No significant difference is seen in ipsilateral hemisphere enlargement at 3 d. (i) At 10 d, CD163^−/− mice have significantly more ICH-induced brain damage. (j) No difference is seen in the amount of hematoidin-pigment (bilirubin) content. At 3 d, comparisons include n = 20 WT and n = 19 CD163^−/− mice. At 10 d, comparisons include n = 11 WT and n = 12 CD163^−/− mice. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 2.

CD163 deficiency temporally influences mortality and functional outcomes after ICH. WT and CD163^−/− mice underwent collagenase-induced ICH and were survived to 10 d. (a) Log-rank analysis demonstrates no difference in Kaplan–Meier survival estimates between groups. (b) However, a temporal disproportion in mortality was identified, where mortality on or before 4 d and after 4 d significantly differed between groups. WT mice account for the majority of deaths on or before 4 d and CD163^−/− mice account for the majority of deaths after 4 d. (c) Regression analysis reveals that WT mice recover neurologic function significantly faster than CD163^−/− mice. (d) Endpoint analyses show that CD163^−/− mice have reduced neurological deficits at 3 d, but greater deficits at 10 d. (e–j) On measures of locomotor activity, regression analyses (e,g,i) show no differences in the rate of recovery between groups, and endpoint analyses (f,h,j) show no differences at 3 d. However, at 10 d, CD163^−/− mice have reduced ambulatory distance and stereotypic time, and increased resting time, while WT mice demonstrate values approximating that of their baseline function. (k,l) On accelerating Rotarod performance, (K) regression analysis shows no difference in the rate of recovery between groups, but at the 3-d endpoint, CD163^−/− mice exhibit improved latency to fall. No differences are seen between groups at the 10-d endpoint. The identified baseline differences in locomotor activity and rotarod performance between groups were statistically accounted for by linear mixed modeling, which also allows estimations of mortality dropouts. P values for regression analyses (c,e,g,i,k) represent slope comparisons. All comparisons include n = 23 WT and n = 17 CD163^−/− mice. *p < 0.05, **p < 0.01.

Figure 3.

CD163 deficiency reduces BBB dysfunction and Hb content. Representative images for WT and CD163^−/− mice showing (a) IgG and (c) Hb immunohistochemistry 3-d post-ICH. Square selections on low-magnification images denote the location of magnified regions. (b) CD163^−/− mice have significantly less BBB dysfunction. (d) CD163^−/− mice display significantly less Hb. Comparisons include n = 7 WT and n = 14 CD163^−/− mice for IgG and n = 5 WT and n = 7 CD163^−/− for Hb. *p < 0.05, **p < 0.01.

Figure 4.

Effect of CD163 deficiency on HO1 and Perls’ iron. Representative images showing HO1 expression for WT and CD163^−/− at (a) 3 d and (e) 10 d and iron deposition at (c) 3 d and (g) 10 d. Square selections on the low magnification images denote the location of magnified regions. HO1 images at 3 d and 10 d were counterstained with Cresyl violet and hematoxylin, respectively. Perls’ iron images were counterstained with nuclear fast red. (b,f) For both endpoints, no difference in HO1 expression is seen. (d) At 3 d, CD163^−/− mice have significantly less iron. (h) At 10 d, CD163^−/− mice have significantly more iron. At 3 d, comparisons include n = 14 WT and n = 16 CD163^−/− mice for Perls’ iron and n = 10 WT and n = 10 CD163^−/− mice for HO1. At 10 d, comparisons include n = 9 WT and n = 12 CD163^−/− mice for Perls’ iron and n = 7 WT and n = 9 CD163^−/− mice for HO1. *p < 0.05, ***p < 0.001.

Figure 5.

CD163 deficiency temporally influences astrogliosis. Representative images showing ipsilateral and contralateral cortical, perihematomal, and hemispheric GFAP immunoreactivity at 3 d for (a) WT and (b) CD163^−/− mice and at 10 d for (f) WT and (g) CD163^−/− mice. Square selections on the low magnification center whole-brain images denote the location of magnified regions. (c–e) At 3 d, CD163^−/− mice demonstrate significantly increased ipsilateral (c) cortical, (d) perihematomal, and (e) hemispheric astrogliosis. (h–j) At 10 d, CD163^−/− mice tend to show reduced astrogliosis in the ipsilateral (h) cortex and (j) hemisphere, but no difference is seen in the (i) perihematomal area. At 3 d, comparisons include n = 5 WT and n = 8 CD163^−/− mice. At 10 d, comparisons include n = 4 WT and n = 6 CD163^−/− mice. *p < 0.05.

Figure 6.

Effect of CD163 deficiency on angiogenesis/neovascularization. Representative images showing ipsilateral cortical and hematomal immunoreactivity of (a) PECAM at 3 d, (d) VEGF at 3 d, and (g) VEGF at 10 d for WT and CD163^−/− mice. Square selections on the low magnification center hemi-brain images denote the location of magnified regions. (b) At 3 d, CD163^−/− mice have significantly increased neovascularization in the motor cortex. (c,e,f) No difference in hematomal neovascularization or cortical and hematomal VEGF expression is seen. (h–i) At 10 d, CD163^−/− mice have increased hematomal VEGF expression, but no difference in cortical expression. At 3 d, comparisons include n = 5 WT and n = 7 CD163^−/− mice for PECAM and n = 7 WT and n = 7 CD163^−/− mice for VEGF. At 10 d, comparisons include n = 6 WT and n = 10 CD163^−/− mice. *p < 0.05.