Long-Term in Vivo Investigation of Mouse Cerebral Microcirculation by Fluorescence Confocal Microscopy in the Area of Focal Ischemia

Abstract

This study was designed to assess that mouse pial and cortical microcirculation can be monitored in the long term directly in the area of focal ischemia, using in vivo fluorescence microscopy. A closed cranial window was placed over the left parieto-occipital cortex of C57BL/6J mice. Local microcirculation was recorded in real time through the window using laser-scanning confocal fluorescence microscopy after intravenous injection of fluorescent erythrocytes and dextran. The basal velocity of erythrocytes through intraparenchymal capillaries was 0.53+/-0.30 mm/sec (n=121 capillaries in 10 mice). Two branches of the middle cerebral artery were topically cauterized through the window. Blood flow evaluated by laser-Doppler flowmetry in two distinct areas indicated the occurrence of an ischemic core (15.2%+/-5.9% of baseline for at least 2 h) and a penumbral zone. Magnetic resonance imaging and histology were used to characterize the ischemic area at 24 h after occlusion. The infarct volume was 7.3+/-3.2 mm(3) (n=6). Microcirculation was repeatedly videorecorded using fluorescence confocal microscopy over the next month. After the decrease following arterial occlusion, capillary erythrocyte velocity was significantly higher than baseline 1 week later, and attained 0.74+/-0.51 mm/sec (n=76 capillaries in six mice, P<0.005) after 1 month, while venous and capillary network remodeling was assessed, with a marked decrease in tortuosity. Immunohistochemistry revealed a zone of necrotic tissue into the infarct epicenter, with activated astrocytes at its border. Such long-term investigations in ischemic cortex brings new insight into the microcirculatory changes induced by focal ischemia and show the feasibility of long-term fluorescence studies in the mouse cortex.

Wide Genetic Variation in the Native Pial Collateral Circulation is a Major Determinant of Variation in Severity of Stroke

“…Diameters of pial collaterals of B6 mice (measured under ketamine and xylazine anesthesia with intravital microscopy through an open cranial window) increase up to fivefold after topical adenosine or sudden increase in shear stress following acute MCAO . Others have reported that pial collaterals exhibit basal tone (Wei et al, 2001;Tomita et al, 2005). Inhibition of substantial basal tone in pial collaterals after MCAO could contribute to the rapid remodeling we observed, as chronic vasodilation is known to stimulate outward remodeling of arterioles (van Gieson et al, 2003).…”

Anesthesia and the Quantitative Evaluation of Neurovascular Coupling

“…For longitudinal experiments, the same anesthetics have been repeatedly used in single animals; i.e., isoflurane (Colonnese et al, 2008;Tomita et al, 2005) and ketamine/xylazine (Brown et al, 2010). For those experiments, good recovery from anesthesia discontinuation is important in the choice of anesthetics for performing controlled experiments.…”

A Direct Method for Measuring Mouse Capillary Cortical Blood Volume Using Multiphoton Laser Scanning Microscopy

“…Direct brain imaging with better spatial (micrometric) and temporal resolutions can be obtained using optical techniques (Helmchen and Denk, 2005). Traditional fluorescence confocal laser scanning microscopy allows an imaging depth of about 200 mm in brain tissue with a high spatial resolution (Tomita et al, 2005) and this imaging depth had been extended with the development of multiphoton laser scanning microscopy, which became the main technique for high-resolution in vivo deep imaging (Denk et al, 1990). A imaging depth of 500-600 mm can be obtained currently in the cortex (Helmchen and Denk, 2005) (this covers around 70% of the mouse cortex) and imaging of brain vessels as deep as 1000 mm has been reported (Theer et al, 2003).…”