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Jesse B. Schallek, Ying Geng, David R. Williams; In Vivo Adaptive Optics Imaging Of Retinal Pericytes And Capillary Blood Velocity In Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6831. doi: https://doi.org/.
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The role of pericytes in actively regulating local capillary blood flow is controversial, in part because of the challenges of imaging these small cells in vivo. The development of adaptive optics retinal imaging combined with cell specific fluorescent labeling in transgenic mice provides the necessary resolution to simultaneously image pericytes and measure capillary blood flow noninvasively.
Adult mice, hemizygous for the NG2 DsRed transgene (Jackson Labs, Bar Harbor, ME), were used to visualize fluorescent retinal pericytes in vivo. Mice were anesthetized with inhaled isoflurane and fitted with a contact lens to prevent corneal drying. Fluorescence adaptive optics scanning laser ophthalmoscopy (FAOSLO) recorded two channels simultaneously. A reflectance channel imaged the vascular network with 789 nm light, revealing individual blood cells moving through the retinal circulation. A second channel captured 579+22 nm DsRed fluorescence excited with a 514 nm laser. Modulation of the excitation light served as a visual stimulus to examine the effects of neurovascular coupling in the mouse retina. Light was flickered at frequencies spanning 5-25 Hz, within the temporal tuning peak of mouse ganglion cells.
We observed a biphasic dilation (~5%) and constriction (~9%) of the central retinal artery when the retina was stimulated with flashing 514nm light. This vascular response to visual stimulation in the mouse retina is consistent with neurovascular coupling reported in other mammals. Adaptive optics imaging also revealed hemostatic events in individual capillaries lasting tens of seconds. Confocal z-stacks acquired with FAOSLO showed the mouse retinal circulation has at least three distinct stratifications, consistent with histological reports. Pericytes showed fluorescence in both somas and processes and co-localized with capillaries at each stratification.
We have imaged for the first time retinal pericyte structure and capillary blood flow simultaneously in the living animal. Large vessel vasomotion in response to visual stimulation shows evidence of intact neurovascular response in the anesthetized mouse. Additionally, the micron-level resolution provided by FAOSLO provides the means for future investigation of the role of pericyte contractility in regulating neurovascular coupling in individual capillaries in the living animal.
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