Abstract

BackgroundFollowing intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to heme and participates in its transport, while heme oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes.MethodsIntracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induce ICH. Hpx knockout (Hpx−/−) or HO2 knockout (HO2−/−) mice were injected with 10 μL of SFHb in the striatum. After injection, behavioral/functional tests were performed, along with anatomical analyses. Iron deposition and neuronal degeneration were depicted by Perls’ and Fluoro-Jade B staining, respectively. Immunohistochemistry with anti-ionized calcium-binding adapter protein 1 (Iba1) was used to estimate activated microglial cells around the injured site.ResultsThis study shows that deleting Hpx or HO2 aggravated SFHb-induced brain injury. Compared to wild-type littermates, larger lesion volumes were observed in Hpx−/− and HO2−/− mice, which also bear more degenerating neurons in the peri-lesion area 24 h postinjection. Fewer Iba1-positive microglial cells were detected at the peri-lesion area in Hpx−/− and HO2−/− mice, interestingly, which is associated with markedly increased iron-positive microglial cells. Moreover, the Iba1-positive microglial cells increased from 24 to 72 h postinjection and were accompanied with improved neurologic deficits in Hpx−/− and HO2−/− mice. These results suggest that Iba1-positive microglial cells could engulf the extracellular SFHb and provide protective effects after ICH. We then treated cultured primary microglial cells with SFHb at low and high concentrations. The results show that microglial cells actively take up the extracellular SFHb. Of interest, we also found that iron overload in microglia significantly reduces the Iba1 expression level and resultantly inhibits microglial phagocytosis.ConclusionsThis study suggests that microglial cells contribute to hemoglobin-heme clearance after ICH; however, the resultant iron overloads in microglia appear to decrease Iba1 expression and to further inhibit microglial phagocytosis.

Highlights

  • Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury

  • Deletion of Hpx and heme oxygenase 2 (HO2) aggravates brain injury after stroma-free hemoglobin (SFHb) injection Our preliminary studies showed that backflow did not occur when mice were injected over a period of 30 min with 10 μL of SFHb

  • Brain sections revealed that the injected 10 μL of SFHb diffused to the whole striatum, which led to behavioral disability

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Summary

Introduction

Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. The hemoglobin and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hpx) contribute to hematoma removal after ICH [12], and Hpx has the highest binding affinity to heme (Kd < 1 pM) [12, 13]. The linear relationship between Hpx concentration and protection defined a highly efficient backup scavenger system during conditions of large excess of free hemoglobin [14]. These together suggest that Hpx could be more critical in hematoma removal after ICH than has been known before. Intraperitoneal injection of bacterial endotoxin has been shown to cause a robust increase in Hp expression in peripheral organs and blood serum, but not in the brain [15]

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