Strokes are the third most common cause of death in the United States. Stroke is caused by occlusion (ischemic stroke) or rupture of cerebral blood vessels (hemorrhagic stroke). Although hemorrhagic strokes comprise less then 20% of all strokes, they are associated with worse mortality and morbidity than ischemic strokes. A better understanding of how brain injury occurs in subarachnoid and intracerebral hemorrhages may suggest alternative therapies. For this reason, the research that comprises this dissertation focuses on brain injury mechanisms in hemorrhagic stroke, with an emphasis on intracerebral hemorrhage (ICH). In these experiments we study two putative mechanisms of injury: (1) the blood degradation products, unconjugated bilirubin (UBR) and bilirubin oxidation products (BOXes), and (2) inflammation. Moreover, we ask whether there is an interaction between these two mechanisms. This question has not been previously addressed in the literature, and it represents a significant contribution of this dissertation.

Chapters 1 and 2 introduce this research by reviewing relevant literature. Before asking what contributions UBR and BOXes have in ICH, we first address gaps in the understanding of how UBR is oxidized to BOXes (Chapter 3). Our data indicate that more than one mechanism exists for conversion of UBR to BOXes. Both cytochrome oxidase and reactive oxygen species produce BOXes in vitro. In Chapter 4, we design a mouse model of intracerebral hemorrhage and determine the profile of inflammatory cell infiltration into the brain after stroke. We found that, compared to saline injection, ICH was associated with an increase in neutrophils at 1 and 4 days and CD4 ⁺ T cells at 4 days (Figures 4.3 and 4.4). In Chapter 5, we show that UBR potentiates inflammation and edema after ICH. We observed an increase in brain water content (77% to 77.5%, p< 0.05, Figure 5.1) when 450 mcM UBR was infused with blood. UBR was associated with increased neutrophil counts assessed by immunofluorescence and flow cytometry (Figures 5.2 and 5.6) and also activated neutrophils in vitro and that this activation was PKC dependent (Figure 5.3). Finally, we found that UBR-treated animals had increased ICAM-1-positive blood vessels (Figure 5.9) and suggest this as a mechanism by which UBR produced an increase in neutrophil inflammation. BOXes were associated with neutrophil activation in vitro (Figure 5.4) but we did not observe changes in inflammation in BOXes-treated mice (Figure 5.8). Chapter 6 asks whether neutrophil depletion improves outcome after ICH. We depleted neutrophils using an antibody to the surface marker GR-1. Compared to control, animals receiving anti-GR-1 had profoundly reduced blood neutrophils (50.3 ± 8.3% vs. 1.5 ± 0.34% of CD45 ⁺ cells, p≤ 0.01, Figure 6.2), but neutrophils in the brain were only reduced by 50% (Figure 6.3). We observed reduced astrocyte reactivity in neutrophil-depleted animals but did not observe changes in behavioral data or brain edema. Taken together, our results suggest that UBR and neutrophils contribute to brain injury after ICH (Chapter 7). More detailed studies that incorporate sensitive behavioral data are needed before suggesting potential therapies.