Acute aneurysmal subarachnoid hemorrhage (aSAH) is a complex and multifaceted disorder that plays out over days to weeks. Because of this relationship between cerebrovasospasm (CV), delayed cerebral ischemia (DCI), and poor SAH outcomes, there have been unsuccessful efforts made to establish treatments that decrease the incidence of CV. Identifying a predisposing genetic factor and/or biomarker for the early prediction of CV would serve as a clinically useful tool in the critical care management of aSAH patients. Knowing that CV peaks at 7-9 days post SAH, we have a unique therapeutic window here–as long as we have the right predictive tools. Following aSAH, hemolysis in the subarachnoid space releases large amounts of free hemoglobin (Hb). This toxic pro-oxidative and pro-inflammatory Hb and metabolites (iron, bilirubin, and bilirubin oxidation products) are then directly in extravascular contact with the main arteries supplying the brain. A few studies have suggested that changes in the Hb concentrations within the subarachnoid space (i.e. CSF) tend to mirror the evolution of CV; although, the mechanisms by which free Hb may cause this delayed CV are poorly understood. Haptoglobin, hemopexin, sCD163, transferrin, and ferritin are some of the main detoxifying binding proteins against toxic free Hb and metabolites. Indeed, we have recently reported in PNAS that haptoglobin 2-2 genotype could be an independent risk factor for CV, DCI, and poor long-term functional SAH outcomes, likely as a result of ineffective management and clearance of free Hb from the subarachnoid space. Here, we plan to test this hypothesis by detailed mechanistic analyses and simultaneously extend these results to prognostic and diagnostic biomarker development using two complementary analytical methods: 1) unbiased broad profiling with an iTRAQ nanoflow LC-MS/MS-based approach, and 2) targeted profiling on a luminex-based Multi-Analyte Profile platform. Inclusion of such proteome profiling approach provides a strong exploratory aspect to this R21 proposal that is most likley to provide additional unbiased novel pathways. Aim 1: To investigate the biomarker potential of toxic hemoglobin and metabolites in predicting key aSAH clinical events. Aim 2: To investigate the biomarker potential of the protective hemoglobin and metabolite- binding proteins in predicting key aSAH clinical events. Paired serum and CSF levels of Hb, metabolites, and protective binding proteins will be measured at admission and at 6h intervals thereafter for up to 14d post- bleed. These temporal profiles will be correlated to the incidence, severity, and dynamics of CV (rise, peak, resolution), incidence of DCI, mortality, and functional outcomes at discharge, 6wk, and 12mo post-bleed. We have already collected the majority of this clinical data in a standardized manner. Together, this approach will allow the potential development of candidate prognostic and diagnostic biomarkers and may provide a better/novel mechanistic understanding of the dynamics of blood clearance after aSAH. As such, we expect to be able to identify the most opportune key players in these pathways for future therapeutic targeting.

Stroke is the primary cause of adult disability, and it accounts for 1 of every 18 deaths and ~800,000 Americans suffer a new or recurrent stroke each year. While both ischemic and hemorrhagic strokes cause serious neurological damage, intracranial bleeding has worse consequences and is associated with significantly more morbidity and mortality. Intraparenchymal hemorrhage and notably subarachnoid hemorrhage (SAH) have not been investigated as much as ischemic stroke. Understanding the clinical underlying neuropathophysiological processes are a necessary first step to begin novel hypothesis-driven preclinical work aimed at developing new stroke therapies. We propose to use our unique collection of banked biospecimens from SAH patients to gain a mechanistic understanding of these processes such that we can in the near future we can potentially predict patients who will have complications and develop novel preventative/therapeutic treatments.