Targeted real time imaging of clot formation and the study of astrocyte injury during ischemia

Abstract

Astrocytes are the predominant glial-cell type of the neurovascular unit but little is known about their functional impact during ischemia. We describe the use of a modified photothrombotic technique employing a tightly focused 532nm green laser to optically excite the circulating photosensitizer rose bengal whilst visualization in real time clot formation and astrocyte injury under two-photon microscopy (2-PTM). Targeted vascular occlusions in small vessels of the brain is an important technique to study the microscopic basis of cerebral ischemia and vascular dementia. Using 2-PTM of green fluorescent protein-labeled astrocytes (GFAP-GFP) and 10-kDa Cascade Blue dextran to label the vasculature, we explore the alteration of the spatial relationship between cortical microcirculation and astrocytic structure in vivo during ischemia. This 2-PTM method allows extremely high spatial and temporal resolution for studying pathological mechanisms that underlie ischemic injury, including abnormal changes in cell signaling and structure, vascular dysfunction, and inflammation. This method ensures that instances of spontaneous clot dissolution and recanalization do not precipitate and confirms that a stable clot is formed and maintained throughout the observation period. We aim to identify the cellular basis of cerebrovascular dysregulation in transgenic animal models of neurological disease to identify molecular targets for the pharmacotherapy of these pathological pathways. By using this technique we could follow in real-time clot formation via a conventional scanner. Tornado bleaching generated a clot via a SIM scanner that was integrated in the light path. To block tissue autofluoresence and reflected light from the green laser, the light path was blocked by a 420-465nm filter that only permitted the blue emission of the intravenous injected cascade blue dextran used to label the plasma. Unlike other methods this technique allows real-time imaging of clot formation and permits the visualization of the green clot thus formed after intravenous loading of rhodamine 6G (0.3mg/kg in 300µL PBS) by switching back to the green channel. Two photon laser scanning fluorescence microscopy permitted the observation of changes in blood flow, blood redistribution after clot formation, platelet aggregation and the loss of integrity of neighbouring astrocytes through a cranial window in GFP-GFAP-expressing mice after targeted occlusion.