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.

Intracerebral hemorrhage (ICH) is a devastating type of stroke characterized by bleeding into the brain parenchyma and secondary brain injury resulting from strong neuroinflammatory responses to blood components. Production of prostaglandin E2 (PGE2) is significantly upregulated following ICH and contributes to this inflammatory response in part through its E prostanoid receptor subtype 2 (EP2). Signaling through the EP2 receptor has been shown to affect outcomes of many acute and chronic neurological disorders; although, not yet explored in the context of ICH. Wildtype (WT) and EP2 receptor knockout (EP2−/−) mice were subjected to ICH, and various anatomical and functional outcomes were assessed by histology and neurobehavioral testing, respectively. When compared with age-matched WT controls, EP2−/− mice had 41.9 ± 4.7% smaller ICH-induced brain lesions and displayed significantly less ipsilateral hemispheric enlargement and incidence of intraventricular hemorrhage. Anatomical outcomes correlated with improved functional recovery as identified by neurological deficit scoring. Histological staining was performed to begin investigating the mechanisms involved in EP2-mediated neurotoxicity after ICH. EP2−/− mice exhibited 45.5 ± 5.8% and 41.4 ± 8.1% less blood and ferric iron accumulation, respectively. Furthermore, significantly less striatal and cortical microgliosis, striatal and cortical astrogliosis, blood–brain barrier breakdown, and peripheral neutrophil infiltration were seen in EP2−/− mice. This study is the first to suggest a deleterious role for the PGE2-EP2 signaling axis in modulating brain injury, inflammation, and functional recovery following ICH. Targeting the EP2 G protein-coupled receptor may represent a new therapeutic avenue for the treatment of hemorrhagic stroke.

Intracerebral hemorrhage (ICH) is a stroke subtype associated with high mortality and morbidity. Following ICH, excitotoxicity and inflammation significantly contribute to secondary brain injury and poor outcomes. Prostaglandin E2 (PGE2) levels rise locally with insult to the nervous system, and PGE2 is known to modulate these processes mainly through its E prostanoid (EP) receptors, EP1–4. EP3 is the most abundant EP receptor in the brain and we have previously shown that signaling through the PGE2-EP3 axis exacerbates excitotoxicity and ischemic stroke outcomes. This study aimed to investigate the contribution of this pathway in modulating anatomical outcomes and functional recovery following ICH. Genetic deletion of the EP3 receptor resulted in 48.2 ± 7.3% less ICH-induced brain injury (p < 0.005) and improved functional recovery (p < 0.05), as identified by neurological deficit scoring. To start investigating the mechanisms involved in neuroprotection with impaired PGE2-EP3 signaling, histological staining was performed to evaluate blood and ferric iron accumulation, neuroinflammation, blood brain barrier dysfunction, and peripheral neutrophil infiltration. After ICH, EP3−/− mice demonstrated 49.5 ± 8.8% and 42.8 ± 13.1% less blood (p < 0.01) and ferric iron content (p < 0.05), respectively. Furthermore, EP3−/− mice had significantly reduced astrogliosis, microglial activation, blood brain barrier breakdown, and neutrophil infiltration. Collectively, these results suggest an injurious role for the PGE2-EP3 signaling axis in modulating brain injury, inflammation, and neurologic functional recovery after ICH. Modulation of the PGE2-EP3 signaling axis may represent a putative therapeutic avenue for the treatment of ICH.

Following intracerebral hemorrhage (ICH), extracellular heme precipitates secondary brain injury, which results in irreversible brain damage and enduring neurological deficits. Hemopexin (Hpx) is an endogenous protein responsible for scavenging heme, thereby modulating its intrinsic proxidant/proinflammatory properties. Although Hpx is present in the brain, the endogenous levels are insufficient to combat the massive heme overload following ICH. We hypothesized that increasing brain Hpx levels would improve ICH outcomes. Unique recombinant adeno-associated viral vectors were designed to specifically overexpress Hpx within the mouse brain. Western blotting, ELISA, and immunohistochemistry of brain homogenates/sections, CSF, and serum were performed. As compared to controls, Hpx mice have increased Hpx protein levels in all three types of biospecimens evaluated, which results in 45.6 ± 6.9% smaller lesions and improved functional recovery after ICH (n=14-19/group, p < 0.05). Local mechanistic analyses show significantly less tissue injury, trends toward smaller hematoma volumes, unchanged heme oxygenase 1 and iron levels, and significantly increased microgliosis and decreased astrogliosis and lipid peroxidation. Peripheral levels of heme-related markers indicate a positive modulation of iron-binding capacity. These findings reveal that high local Hpx levels improve ICH outcomes, likely through both central and peripheral clearance mechanisms, and establish the potential for therapeutically administering clinical-grade Hpx for ICH.

With the population aging at an accelerated rate, the prevalence of stroke and financial burden of stroke-related health care costs are expected to continue to increase. Intracerebral hemorrhage (ICH) is a devastating stroke subtype more commonly affecting the elderly population, who display increased mortality and worse functional outcomes compared with younger patients. This study aimed to investigate the contribution of the prostaglandin E2 (PGE2) E prostanoid (EP) receptor subtype 3 in modulating anatomical outcomes and functional recovery following ICH in 24-mo-old mice. EP3 is the most abundant EP receptor in the brain and we have previously shown that signaling through the PGE2-EP3 axis exacerbates ICH outcomes in young mice. Here, we show that EP3 receptor deletion results in 17.9 ± 6.1% less ICH-induced brain injury (P < 0.05) and improves neurological functional recovery (P < 0.01), as identified by lower neurological deficit scores, decreased resting time, and more gross and fine motor movements. Immunohistological staining was performed to investigate possible mechanisms of EP3-mediated neurotoxicity. Identified mechanisms include reduced blood accumulation and modulation of angiogenic and astroglial responses. Using this aged cohort of mice, we have confirmed and extended our previous results in young mice demonstrating the deleterious role of the PGE2-EP3 signaling axis in modulating brain injury and functional recovery after ICH, further supporting the notion of the EP3 receptor as a putative therapeutic avenue for the treatment of ICH.