Haptoglobin phenotype predicts the development of focal and global cerebral vasospasm and may influence outcomes after aneurysmal subarachnoid hemorrhage.

Cerebral vasospasm (CV) and the resulting delayed cerebral ischemia (DCI) significantly contribute to poor outcomes following aneurysmal subarachnoid hemorrhage (aSAH). Free hemoglobin (Hb) within the subarachnoid space has been implicated in the pathogenesis of CV. Haptoglobin (Hp) binds free pro-oxidant Hb, thereby modulating its harmful effects. Humans can be of three Hp phenotypes: Hp1-1, Hp2-1, or Hp2-2. In several disease states, the Hp2-2 protein has been associated with reduced ability to protect against toxic free Hb. We hypothesized that individuals with the Hp2-2 phenotype would have more CV, DCI, mortality, and worse functional outcomes after aSAH. In a sample of 74 aSAH patients, Hp2-2 phenotype was significantly associated with increased focal moderate (P = 0.014) and severe (P = 0.008) CV and more global CV (P = 0.014) after controlling for covariates. Strong trends toward increased mortality (P = 0.079) and worse functional outcomes were seen for the Hp2-2 patients with modified Rankin scale at 6 wk (P = 0.076) and at 1 y (P = 0.051) and with Glasgow Outcome Scale Extended at discharge (P = 0.091) and at 1 y (P = 0.055). In conclusion, Hp2-2 phenotype is an independent risk factor for the development of both focal and global CV and also predicts poor functional outcomes and mortality after aSAH. Hp phenotyping may serve as a clinically useful tool in the critical care management of aSAH patients by allowing for early prediction of those patients who require increased vigilance due to their inherent genetic risk for the development of CV and resulting DCI and poor outcomes.

PGE2-EP3 signaling exacerbates intracerebral hemorrhage outcomes in 24-mo-old mice.

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.

Genetic deletion of the prostaglandin E2 E prostanoid receptor subtype 3 improves anatomical and functional outcomes after intracerebral hemorrhage.

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. EP receptor subtype 3 (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 EP3 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 knockout mice demonstrated 49.5 ± 8.8% and 42.8 ± 13.1% less blood (P < 0.01) and ferric iron (P < 0.05), respectively. Furthermore, EP3 knockout 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 neurological functional recovery after ICH. Modulation of the PGE2 -EP3 signaling axis may represent a putative therapeutic avenue for the treatment of ICH.

Intracerebral hemorrhage outcomes following selective blockade or stimulation of the PGE2 EP1 receptor.

BACKGROUND: Inflammation following intracerebral hemorrhage (ICH) significantly contributes to secondary brain damage and poor outcomes. Prostaglandin E2 (PGE2) is known to modulate neuroinflammatory responses and is upregulated in response to brain injury as a result of changes in inducible cyclooxygenase 2 (COX-2) and the membrane-bound type of PGE synthase. Inhibition of COX-2 activity has been reported to attenuate ICH-induced brain injury; however, the clinical utility of such drugs is limited due to the potential for severe side effects. Therefore, it is now important to search for downstream targets capable of preferentially modulating PGE2 signaling, and the four E prostanoid receptors, EP1-4, which are the main targets of PGE2, remain a viable therapeutic option. We have previously shown that EP1 receptor deletion aggravates ICH-induced brain injury and impairs functional recovery, thus the current study aimed to elaborate on these results by including a pharmacologic approach targeting the EP1 receptor. RESULTS: Chronic post-treatment with the selective EP1 receptor antagonist, SC-51089, increased lesion volume by 30.1 ± 14.5% (p < 0.05) and treatment with the EP1 agonist, 17-pt-PGE2, improved neuromuscular functional recovery on grip strength (p < 0.01) and hanging wire (p < 0.05) behavioral testing. To begin identifying the mechanisms involved in EP1-mediated neuroprotection after ICH, histology was performed to assess ferric iron content, neuroinflammation, leukocyte transendothelial migratory potential, and peripheral neutrophil and immunoglobulin infiltration. Following ICH, mice treated with the antagonist displayed increased ferric iron (p < 0.05) and cortical microgliosis (p < 0.05), whereas treatment with the agonist decreased cortical (p < 0.01) and striatal (p < 0.001) astrogliosis, leukocyte transendothelial migratory potential (p < 0.01), neutrophil infiltration (p < 0.05), and blood brain barrier breakdown (p < 0.05). CONCLUSIONS: In agreement with our previous results, selective antagonism of the EP1 receptor aggravated ICH-induced brain injury. Furthermore, EP1 receptor agonism improved anatomical outcomes and functional recovery. Thus, the present data continues to reinforce a putative role for EP1 as a new and more selective therapeutic target for the treatment of ICH that could reduce the side effects associated with COX-2 inhibition while still exploiting the beneficial effects.

Role of the PGE2 receptor subtypes EP1, EP2, and EP3 in repetitive traumatic brain injury.

AIMS: The goal was to explore the signaling pathways of PGE2 to investigate therapeutic effects against secondary injuries following TBI. METHODS: Young (4.9 ± 1.0 months) and aged (20.4 ± 1.4 months) male wild type (WT) C57BL/6 and PGE2 EP1, 2, and 3 receptor knockout mice were selected to either receive sham or repetitive concussive head injury. Immunohistochemistry protocols with Iba1 and GFAP were performed to evaluate microgliosis and astrogliosis in the hippocampus, two critical components of neuroinflammation. Passive avoidance test measured memory function associated with the hippocampus. RESULTS: No differences in hippocampal microgliosis were found when aged EP2-/- and EP3-/- mice were compared with aged WT mice. However, the aged EP1-/- mice had 69.2 ± 7.5% less hippocampal microgliosis in the contralateral hemisphere compared with WT aged mice. Compared with aged EP2-/- and EP3-/- , EP1-/- aged mice had 78.9 ± 5.1% and 74.7 ± 6.2% less hippocampal microgliosis in the contralateral hemisphere. Within the EP1-/- mice, aged mice had 90.7 ± 2.7% and 81.1 ± 5.6% less hippocampal microgliosis compared with EP1-/- young mice in the contralateral and ipsilateral hemispheres, respectively. No differences were noted in all groups for astrogliosis. There was a significant difference in latency time within EP1-/- , EP2-/- , and EP3-/- on day 1 and day 2 in aged and young mice. CONCLUSION: These findings demonstrate that the PGE2 EP receptors may be potential therapeutic targets to treat repetitive concussions and other acute brain injuries.

Effect of Experimental Ischemic Stroke and PGE2 EP1 Selective Antagonism in Alzheimer's Disease Mouse Models.

BACKGROUND: Neuroinflammation has been recognized as an important factor in the pathogenesis of Alzheimer's disease (AD). One of the most recognized pathways in mediating neuroinflammation is the prostaglandin E2-EP1 receptor pathway. OBJECTIVE: Here, we examined the efficacy of the selective EP1 antagonist ONO-8713 in limiting amyloid-ß (Aß), lesion volumes, and behavioral indexes in AD mouse models after ischemic stroke. METHODS: Transgenic APP/PS1, 3xTgAD, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. Functional outcomes, memory, anatomical outcomes, and Aß concentrations were assessed 14 days after surgery. RESULTS: pdMCAO resulted in significant deterioration in functional and anatomical outcomes in the transgenic mice compared with the WT mice. No relevant differences were observed in the behavioral tests when comparing the ONO-8713 and vehicle-treated groups. Significantly lower cavitation (p = 0.0373) and percent tissue loss (p = 0.0247) were observed in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 mice. However, the percent tissue injury was significantly higher in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 group (p = 0.0373). Percent tissue loss was also significantly lower in the 3xTgAD + ONO-8713 mice than in the WT + ONO-8713 mice (p = 0.0185). ONO-8713 treatment also attenuated cortical microgliosis in APP/PS1 mice as compared with the vehicle (p = 0.0079); however, no differences were observed in astrogliosis across the groups. Finally, APP/PS1 mice presented with characteristic Aß load in the cortex while 3xTgAD mice exhibited very low Aß levels. CONCLUSION: In conclusion, under the experimental conditions, EP1 receptor antagonist ONO-8713 showed modest benefits in anatomical outcomes after stroke, mainly in APP/PS1 mice.

Temporal and age-dependent effects of haptoglobin deletion on intracerebral hemorrhage-induced brain damage and neurobehavioral outcomes.

Intracerebral hemorrhage (ICH) is a devastating stroke subtype and the presence of extracorpuscular hemoglobin (Hb) exacerbates brain damage. Haptoglobin (Hp) binds Hb, which prevents its oxidation and participation in neurotoxic reactions. Multiple studies have investigated the role of Hp under conditions of intravascular hemolysis, but little is known about its role in the brain and following ICH where extravascular hemolysis is rampant. Young and aged wildtype and Hp-/- mice underwent the autologous blood or collagenase ICH model. Early after ICH, Hp-/- mice display 58.0 ±â€¯5.6% and 36.7 ±â€¯6.9% less brain damage in the autologous blood and collagenase ICH models, respectively. In line with these findings, Hp-/- mice display less neurological deficits on several neurobehavioral tests. Hp-/- mice have less Perl's iron content, HO1 expression, and blood brain barrier dysfunction, but no difference in brain Hb content, astrogliosis and angiogenesis/neovascularization. At the later endpoint, the young cohort displays 27.8 ±â€¯9.3% less brain damage, while no difference is seen with the aged cohort. For both cohorts, no differences are seen in HO1 levels or iron accumulation, but young Hp-/- mice display less thalamic astrogliosis and striatal microgliosis. This study reveals that the presence or absence of Hp exerts important time- and age-dependent influences on ICH outcomes.

Haptoglobin genotype and aneurysmal subarachnoid hemorrhage: Individual patient data analysis.

OBJECTIVE: To perform an individual patient-level data (IPLD) analysis and to determine the relationship between haptoglobin (HP) genotype and outcomes after aneurysmal subarachnoid hemorrhage (aSAH). METHODS: The primary outcome was favorable outcome on the modified Rankin Scale or Glasgow Outcome Scale up to 12 months after ictus. The secondary outcomes were occurrence of delayed ischemic neurologic deficit, radiologic infarction, angiographic vasospasm, and transcranial Doppler evidence of vasospasm. World Federation of Neurological Surgeons (WFNS) scale, Fisher grade, age, and aneurysmal treatment modality were covariates for both primary and secondary outcomes. As preplanned, a 2-stage IPLD analysis was conducted, followed by these sensitivity analyses: (1) unadjusted; (2) exclusion of unpublished studies; (3) all permutations of HP genotypes; (4) sliding dichotomy; (5) ordinal regression; (6) 1-stage analysis; (7) exclusion of studies not in Hardy-Weinberg equilibrium (HWE); (8) inclusion of studies without the essential covariates; (9) inclusion of additional covariates; and (10) including only covariates significant in univariate analysis. RESULTS: Eleven studies (5 published, 6 unpublished) totaling 939 patients were included. Overall, the study population was in HWE. Follow-up times were 1, 3, and 6 months for 355, 516, and 438 patients. HP genotype was not associated with any primary or secondary outcome. No trends were observed. When taken through the same analysis, higher age and WFNS scale were associated with an unfavorable outcome as expected. CONCLUSION: This comprehensive IPLD analysis, carefully controlling for covariates, refutes previous studies showing that HP1-1 associates with better outcome after aSAH.

A Comparison of Pathophysiology in Humans and Rodent Models of Subarachnoid Hemorrhage.

Non-traumatic subarachnoid hemorrhage (SAH) affects an estimated 30,000 people each year in the United States, with an overall mortality of ~30%. Most cases of SAH result from a ruptured intracranial aneurysm, require long hospital stays, and result in significant disability and high fatality. Early brain injury (EBI) and delayed cerebral vasospasm (CV) have been implicated as leading causes of morbidity and mortality in these patients, necessitating intense focus on developing preclinical animal models that replicate clinical SAH complete with delayed CV. Despite the variety of animal models currently available, translation of findings from rodent models to clinical trials has proven especially difficult. While the explanation for this lack of translation is unclear, possibilities include the lack of standardized practices and poor replication of human pathophysiology, such as delayed cerebral vasospasm and ischemia, in rodent models of SAH. In this review, we summarize the different approaches to simulating SAH in rodents, in particular elucidating the key pathophysiology of the various methods and models. Ultimately, we suggest the development of standardized model of rodent SAH that better replicates human pathophysiology for moving forward with translational research.

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

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.

Increased brain hemopexin levels improve outcomes after intracerebral hemorrhage.

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.

Role of Interleukin-10 in Acute Brain Injuries.

Interleukin-10 (IL-10) is an important anti-inflammatory cytokine expressed in response to brain injury, where it facilitates the resolution of inflammatory cascades, which if prolonged causes secondary brain damage. Here, we comprehensively review the current knowledge regarding the role of IL-10 in modulating outcomes following acute brain injury, including traumatic brain injury (TBI) and the various stroke subtypes. The vascular endothelium is closely tied to the pathophysiology of these neurological disorders and research has demonstrated clear vascular endothelial protective properties for IL-10. In vitro and in vivo models of ischemic stroke have convincingly directly and indirectly shown IL-10-mediated neuroprotection; although clinically, the role of IL-10 in predicting risk and outcomes is less clear. Comparatively, conclusive studies investigating the contribution of IL-10 in subarachnoid hemorrhage are lacking. Weak indirect evidence supporting the protective role of IL-10 in preclinical models of intracerebral hemorrhage exists; however, in the limited number of clinical studies, higher IL-10 levels seen post-ictus have been associated with worse outcomes. Similarly, preclinical TBI models have suggested a neuroprotective role for IL-10; although, controversy exists among the several clinical studies. In summary, while IL-10 is consistently elevated following acute brain injury, the effect of IL-10 appears to be pathology dependent, and preclinical and clinical studies often paradoxically yield opposite results. The pronounced and potent effects of IL-10 in the resolution of inflammation and inconsistency in the literature regarding the contribution of IL-10 in the setting of acute brain injury warrant further rigorously controlled and targeted investigation.

Prostaglandin E2 EP2 receptor deletion attenuates intracerebral hemorrhage-induced brain injury and improves functional recovery.

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.

Detrimental role of the EP1 prostanoid receptor in blood-brain barrier damage following experimental ischemic stroke.

Cyclooxygenase-2 (COX-2) is activated in response to ischemia and significantly contributes to the neuroinflammatory process. Accumulation of COX-2-derived prostaglandin E2 (PGE2) parallels the substantial increase in stroke-mediated blood-brain barrier (BBB) breakdown. Disruption of the BBB is a serious consequence of ischemic stroke, and is mainly mediated by matrix metalloproteinases (MMPs). This study aimed to investigate the role of PGE2 EP1 receptor in neurovascular injury in stroke. We hypothesized that pharmacological blockade or genetic deletion of EP1 protects against BBB damage and hemorrhagic transformation by decreasing the levels and activity of MMP-3 and MMP-9. We found that post-ischemic treatment with the EP1 antagonist, SC-51089, or EP1 genetic deletion results in a significant reduction in BBB disruption and reduced hemorrhagic transformation in an experimental model of transient focal cerebral ischemia. These neurovascular protective effects of EP1 inactivation are associated with a significant reduction in MMP-9/-3, less peripheral neutrophil infiltration, and a preservation of tight junction proteins (ZO-1 and occludin) composing the BBB. Our study identifies the EP1 signaling pathway as an important link between neuroinflammation and MMP-mediated BBB breakdown in ischemic stroke. Targeting the EP1 receptor could represent a novel approach to diminish the devastating consequences of stroke-induced neurovascular damage.

Clinical Implications of Bilirubin-Associated Neuroprotection and Neurotoxicity.

Bilirubin is a primary product of heme catabolism and exhibits both neuroprotective and neurotoxic effects. When present at physiologic concentrations, bilirubin is a potent antioxidant and serves to protect brain tissue from oxidative stress insults. The use of the anesthetic propofol attenuates ischemic injury in rats by exploiting these neuroprotective properties. At pathologic levels, bilirubin has been implicated as a neurotoxic agent, demonstrating the ability to aggregate and adhere to cellular membranes, thereby disrupting normal cellular function. Bilirubin-associated toxicities are amplified by administering drugs such as anesthetics that compete with bilirubin for albumin binding sites, resulting in increased plasma bilirubin concentrations. As such, it is crucial that bilirubin is considered in the critical care management of patients with hemorrhagic stroke, cerebral ischemic damage, and critically ill newborns.