Thesis (M.S.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 40-51). / ix, 51 leaves, bound ill. (some col.) 29 cm

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Persistent cerebrovascular dysfunction has been postulated as one mechanism that may contribute to divergent functional trajectories after ischemic stroke. However, how brain endothelial cells (BECs) acutely respond to ischemia and what endogenous signals subsequently regulate vascular normalization remain poorly understood. To spatiotemporally interrogate neuronal activity and hemodynamics in the acute period after ischemic brain injury we used wide-field imaging. Local ischemia consistently provoked a large-amplitude cortical spreading depolarization (CSD) accompanied by strong vasoconstriction, followed by subsequent diverse CSDs with varying hemodynamic responses. Small CSDs with slow depolarization induced vasodilation in well oxygenated cortical tissue. CSDs of larger amplitude with non-sustained depolarization induced biphasic vascular responses. CSDs of large amplitude, characterized by rapid and prolonged depolarization, drove vasoconstriction in deoxygenated cortical tissue with sustained neuronal depolarization. These observations support a model in which vascular responses after acute brain injury are dependent upon the local relationship between CSD features (i.e. slope, duration, and amplitude of depolarization) and the underlying cortical state (i.e. neuronal activity, perfusion, oxygenation). After this acute period, the ischemic brain is characterized by profound changes in immune cell composition and function. To understand how distinct immune signaling pathways regulate blood-brain barrier (BBB) repair and vascular remodeling after ischemic brain injury, I investigated a unique post-ischemic BEC type one interferon (IFN1) signature. Functional assays and single-cell transcriptomic analyses in IFN1 receptor (Ifnar1) inducible EC knockout (iECKO) mice revealed that loss of BEC IFN1 signaling exacerbated post-stroke barrier disruption and resulted in an expansion of BECs enriched in genes involved in angiogenic processes. Conversely, acute administration of exogenous IFNI ameliorated post stroke BBB disruption. In vitro assays supported that IFNI signaling modulates BEC junctional protein stabilization and vascular endothelial growth factor (VEGF) signaling to enhance BEC barrier properties and suppress angiogenic features, respectively. These findings suggest that endogenous BEC IFN signaling after ischemic brain injury restricts angiogenesis to potentially promote acute barrier function. These studies, which span from the systems to molecular level, demonstrate that brain ischemia and post-ischemic sequelae have a profound impact on cerebrovascular dysfunction and recovery. Furthermore, each study introduces a novel framework to investigate how differences in acute BEC responses may contribute to variable vascular trajectories and longitudinal brain function after ischemic insult.