Abstract
The brain has a high energy demand but little to no energy stores. Therefore, proper brain function relies on the delivery of glucose and oxygen by the cerebral vasculature. The regulation of cerebral blood flow (CBF) occurs at the level of the cerebral capillaries and is driven by a fast and efficient crosstalk between neurons and vessels, a process termed neurovascular coupling (NVC). Experimentally NVC is mainly triggered by sensory stimulation and assessed by measuring either CBF by laser Doppler fluxmetry, laser speckle contrast imaging (LSCI), intrinsic optical imaging, BOLD fMRI, near infrared spectroscopy (NIRS) or functional ultrasound imaging (fUS). Since these techniques have relatively low spatial resolution, diameters of cerebral vessels are mainly assessed by 2-photon microscopy (2-PM). Results of studies on NVC rely on stable animal physiology, high-quality data acquisition, and unbiased data analysis, criteria, which are not easy to achieve. In the current study, we assessed NVC using two different imaging modalities, i.e., LSCI and 2-PM, and analyzed our data using an investigator-independent Matlab-based analysis tool, after manually defining the area of analysis in LSCI and vessels to measure in 2-PM. By investigating NVC in 6–8 weeks, 1-, and 2-year-old mice, we found that NVC was maximal in 1-year old mice and was significantly reduced in aged mice. These findings suggest that NVC is differently affected during the aging process. Most interestingly, specifically pial arterioles, seem to be distinctly affected by the aging. The main finding of our study is that the automated analysis tool works very efficiently in terms of time and accuracy. In fact, the tool reduces the analysis time of one animal from approximately 23 h to about 2 s while basically making no mistakes. In summary, we developed an experimental workflow, which allows us to reliably measure NVC with high spatial and temporal resolution in young and aged mice and to analyze these data in an investigator-independent manner.
Highlights
Since the brain stores very little energy, proper neuronal function depends on a constant supply of glucose and oxygen via cerebral blood flow
We developed a fast, reliable, and unbiased data processing tool for the analysis of neurovascular reactivity (NVC and CO2 inhalation) assessed by laser speckle contrast imaging (LSCI) and 2-photon microscopy (2-PM)
We developed two Matlab-based software tools, the “neurovascular coupling (NVC)-ToolBox,” to analyze neurovascular reactivity by in vivo LSCI and 2-PM imaging following whisker stimulation and hypercapnia
Summary
Since the brain stores very little energy, proper neuronal function depends on a constant supply of glucose and oxygen via cerebral blood flow. The need for nutrients increases and the necessary excess energy is delivered via a tightly regulated redistribution of blood flow to these active areas by dilation of cerebral blood vessels. To investigate NVC in vivo, three different steps are needed: [1] a stimulation paradigm, [2] a fast technique to measure CBF or to visualize cerebral vessel reactivity, and [3] an unbiased and reliable method for data analysis. The software provided with commercially available LSCI units has mainly been developed for data acquisition in single human subjects and has only limited analysis capabilities. This is true when it comes to the analysis of data sets acquired at different time points and in whole groups of subjects. The main aim of the current study is to present a novel tool for the analysis of LSCI and 2-PM data to establish a flexible, software-based analysis pipeline for an automated and investigator-independent analysis of the obtained data sets
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