Lindvere Liis, Dorr Adrienne, Stefanovic Bojana
Sunnybrook Research Institute, Toronto, Ontario M4N 3M5, Canada.
Cold Spring Harb Protoc. 2010 Sep 1;2010(9):pdb.prot5494. doi: 10.1101/pdb.prot5494.
Under physiological conditions, neuronal activity is tightly coupled to hemodynamics of the surrounding microvessels. Conversely, most brain diseases are associated with a disturbance in neurovascular coupling. Measuring the hemodynamic response of the microvascular network to neuronal stimulation in vivo involves two major challenges: maintaining a stable systemic physiological state in the animal and imaging with sufficient temporal and spatial resolution to capture the hemodynamic changes across the three-dimensional cortical microvascular network. Two-photon fluorescence microscopy allows imaging of intact cortex in situ at micrometer spatial and microsecond temporal resolution. However, this modality necessitates focal opening of the skull because of its high scattering of light waves. This protocol describes in detail the requisite surgical preparation and physiological maintenance of an adolescent rat with a closed cranial window. Two-photon fluorescence laser scanning microscopy is then used to image the hemodynamic response of the microvasculature of the primary somatosensory cortex to electrical stimulation of the forepaw, with the end goal of quantitative analysis of brain hemodynamics.
在生理条件下，神经元活动与周围微血管的血流动力学紧密耦合。相反，大多数脑部疾病都与神经血管耦合紊乱有关。在体内测量微血管网络对神经元刺激的血流动力学反应涉及两个主要挑战：在动物体内维持稳定的全身生理状态，以及以足够的时间和空间分辨率进行成像，以捕捉三维皮质微血管网络中的血流动力学变化。双光子荧光显微镜能够以微米级空间分辨率和微秒级时间分辨率对完整的原位皮质进行成像。然而，由于光波的高散射，这种成像方式需要对头骨进行局部开孔。本方案详细描述了具有封闭颅窗的青春期大鼠所需的手术准备和生理维持。然后使用双光子荧光激光扫描显微镜对初级体感皮层微血管对前爪电刺激的血流动力学反应进行成像，最终目标是对脑血流动力学进行定量分析。
