The Role of Hemoglobin Oxidation Products in Triggering Inflammatory Response Upon Intraventricular Hemorrhage in Premature Infants.

Intraventricular hemorrhage (IVH) is a frequent complication of prematurity that is associated with high neonatal mortality and morbidity. IVH is accompanied by red blood cell (RBC) lysis, hemoglobin (Hb) oxidation, and sterile inflammation. Here we investigated whether extracellular Hb, metHb, ferrylHb, and heme contribute to the inflammatory response after IVH. We collected cerebrospinal fluid (CSF) (n = 20) from premature infants with grade III IVH at different time points after the onset of IVH. Levels of Hb, metHb, total heme, and free heme were the highest in CSF samples obtained between days 0 and 20 after the onset of IVH and were mostly non-detectable in CSF collected between days 41 and 60 of post-IVH. Besides Hb monomers, we detected cross-linked Hb dimers and tetramers in post-IVH CSF samples obtained in days 0-20 and 21-40, but only Hb tetramers were present in CSF samples obtained after 41-60 days. Vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) levels were higher in CSF samples obtained between days 0 and 20 than in CSF collected between days 41 and 60 of post-IVH. Concentrations of VCAM-1, intercellular adhesion molecule-1 (ICAM-1), and IL-8 strongly correlated with total heme levels in CSF. Applying the identified heme sources on human brain microvascular endothelial cells revealed that Hb oxidation products and free heme contribute to the inflammatory response. We concluded that RBC lysis, Hb oxidation, and heme release are important components of the inflammatory response in IVH. Pharmacological interventions targeting cell-free Hb, Hb oxidation products, and free heme could have potential to limit the neuroinflammatory response following IVH.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

RATIONALE: Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. OBJECTIVE: To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. METHODS AND RESULTS: Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. CONCLUSIONS: For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

Heme-Hemopexin Scavenging Is Active in the Brain and Associates With Outcome After Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Long-term outcome after subarachnoid hemorrhage (SAH) is potentially linked to cytotoxic heme. Free heme is bound by hemopexin and rapidly scavenged by CD91. We hypothesized that heme scavenging in the brain would be associated with outcome after hemorrhage. METHODS: Using cerebrospinal fluid and tissue from patients with SAH and control individuals, the activity of the intracranial CD91-hemopexin system was examined using ELISA, ultrahigh performance liquid chromatography, and immunohistochemistry. RESULTS: In control individuals, cerebrospinal fluid hemopexin was mainly synthesized intrathecally. After SAH, cerebrospinal fluid hemopexin was high in one third of cases, and these patients had a higher probability of delayed cerebral ischemia and poorer neurological outcome. The intracranial CD91-hemopexin system was active after SAH because CD91 positively correlated with iron deposition in brain tissue. Heme-hemopexin uptake saturated rapidly after SAH because bound heme accumulated early in the cerebrospinal fluid. When the blood-brain barrier was compromised after SAH, serum hemopexin level was lower, suggesting heme transfer to the circulation for peripheral CD91 scavenging. CONCLUSIONS: The CD91-heme-hemopexin scavenging system is important after SAH and merits further study as a potential prognostic marker and therapeutic target.

The pathology of superficial siderosis of the central nervous system.

Chronic or intermittent extravasations of blood into the subarachnoid space, and dissemination of heme by circulating cerebrospinal fluid, are the only established causes of superficial siderosis of the central nervous system (CNS). We studied the autopsy tissues of nine patients by iron histochemistry, immunocytochemistry, single- and double-label immunofluorescence, electron microscopy of ferritin, and high-definition X-ray fluorescence. In one case, frozen brain tissue was available for quantitative assay of total iron and ferritin. Siderotic tissues showed extensive deposits of iron and ferritin, and infiltration of the cerebellar cortex was especially severe. In addition to perivascular collections of hemosiderin-laden macrophages, affected tissues displayed iron-positive anuclear foamy structures in the neuropil that resembled axonal spheroids. They were especially abundant in eighth cranial nerves and spinal cord. Double-label immunofluorescence of the foamy structures showed co-localization of neurofilament protein and ferritin but comparable merged images of myelin-basic protein and ferritin, and ultrastructural visualization of ferritin, did not allow the conclusion that axonopathy was simply due to dilatation and rupture of fibers. Heme-oxygenase-1 (HO-1) immunoreactivity persisted in macrophages of siderotic cerebellar folia. Siderosis caused a large increase in total CNS iron but high-definition X-ray fluorescence of embedded tissue blocks excluded the accumulation of other metals. Holoferritin levels greatly exceeded the degree of iron accumulation. The susceptibility of the cerebellar cortex is likely due to Bergmann glia that serve as conduits for heme; and the abundance of microglia. Both cell types biosynthesize HO-1 and ferritin in response to heme. The eighth cranial nerves are susceptible because they consist of CNS axons, myelin, and neuroglial tissue along their subarachnoid course. The persistence of HO-1 protein implies continuous exposure of CNS to free heme or an excessively sensitive transcriptional response of the HO-1 gene. The conversion of heme iron to hemosiderin probably involves both translational and transcriptional activation of ferritin biosynthesis.

Cerebrospinal fluid ferritin in chronic hydrocephalus after aneurysmal subarachnoid hemorrhage.

OBJECTIVES: Subarachnoid hemorrhage (SAH) is a common cause of chronic hydrocephalus. Blood in the subarachnoid space is intracranially metabolized to bilirubin and iron, and free iron is thereafter detoxified by ferritin. However, no studies have reported the relationship between intracranial heme metabolism and chronic hydrocephalus after SAH. The goal of this prospective study was to clarify the relationship between intracranial heme metabolism and chronic hydrocephalus after SAH. METHODS: The authors measured the levels of bilirubin, iron and ferritin in the cerebrospinal fluid (CSF) of 70 consecutive patients with aneurysmal SAH of Fisher computed tomography Group III, and determined the relationship between these substances' levels and hydrocephalus requiring ventriculoperitoneal shunting. RESULTS: The CSF concentrations of ferritin and inflammatory cells were significantly higher in shunted patients (n = 27) than in non-shunted patients (n = 43) on Days 3 and 4 (p < 0.05 in ferritin and p < 0.01 in inflammatory cells) and 11 to 14 (p < 0.005 in ferritin) post-SAH. These results were independent of other clinical factors. The occurrence of chronic hydrocephalus was not affected by the extent of the intracranial heme metabolism in terms of the bilirubin and iron levels. CONCLUSIONS: This is the first study to show that patients who subsequently had chronic hydrocephalus requiring CSF shunting were associated with higher CSF levels of ferritin in the acute stage of SAH. Higher CSF ferritin levels may not reflect the amount of blood in the subarachnoid space that was intracranially metabolized, but rather more intense subarachnoid inflammatory reactions which may cause chronic hydrocephalus after SAH.