Neurovascular Reactivity in the Aging Mouse Brain Assessed by Laser Speckle Contrast Imaging and 2-Photon Microscopy: Quantification by an Investigator-Independent Analysis Tool

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.

Keywords: aging; hypercapnia; investigator-independent analysis; laser speckle contrast imaging; neurovascular coupling; two-photon microscopy.

Figure 1

Assessment of neurovascular coupling (NVC) using laser speckle contrast imaging (LSCI) and a Matlab-based analysis pipeline. (A) Experimental setup for whisker stimulation and data analysis. Data visualization with LSCI shows a flow map and a perfusion trace (left), data processing steps show binary data exportation, ROI alignment, segmentation, % change map, grid preparation for quantification, and final averaged heat map (right). (B) Heat maps of cerebral perfusion (CBF) in individual mice of different ages following whisker stimulation. Each perfusion map was created by averaging LSCI values from three whisker stimulations. Mean depicts the average of all animals in one group. The dark blue color indicates no or small changes in cortical perfusion, while the yellow color indicates increases of cortical perfusion of up to 15%. (C) Quantification of maximal CBF changes. Young and 1-year-old mice had significantly higher NVC responses in comparison to 2 years old mice (*P < 0.05: 6–8 weeks vs. 2 years old, ***P < 0.001: 1 year vs. 2 year, One-way ANOVA). (D) Quantification of the velocity of CBF increase after whisker stimulation by slope analysis. Two year old mice had a significantly slower CBF increase in comparison to young and 1 year old mice (*P < 0.05: 6–8 weeks vs. 2 year and 1 year vs. 2 year) (n = 6–10 mice/group, mean ± SEM, One-way ANOVA).

Figure 2

Assessment of CO₂ reactivity using laser speckle contrast imaging (LSCI). (A) Experimental setup of the CO₂ challenge (left) and exemplary traces for LSCI perfusion (top right) and end-tidal CO₂ (bottom right). (B) CBF changes before, during, and after hypercapnia in the three investigated age groups. Two-year-old mice showed a low (C) and slow (D) CBF increase during hypercapnia, whereas young and 1-year-old mice had higher peak values (*P < 0.05: 6–8 weeks vs. 2 year), and faster slope changes (*P < 0.05: 6–8 weeks vs. 2 year and 1 year vs. 2 year). (E) Quantification of the area under the curve (AUC) of graph B depicts smaller AUC in 2 year old mice in comparison to 6–8 weeks and 1 year old mice (*P < 0.05) (mean ± SEM, n = 6–8 mice/group, One-way ANOVA).

Figure 3

Assessment of vessel diameter by 2-photon microscopy. (A) Experimental setup for whisker stimulation (top left) and hypercapnia during (top right) and a schematic drawing of the data analysis work flow by the newly developed Matlab script. (B) Quantification of maximal diameter changes in different vessel segments at different ages following whisker stimulation. Pial vessel reactivity increased with age (*P < 0.05: 6–8 weeks vs. 1 year and ***P < 0.001: 6–8 week vs. 2 year) while over all the capillary response was reduced in 2-year-old mice in comparison to young and 1-year-old mice. (C) Quantification of maximal diameter changes in different vessel segments at different ages following hypercapnia. Neurovascular reactivity of cerebral capillaries was impaired in 2-year-old mice as compared to 6–8 weeks and 1-year-old mice (*P < 0.05: 1 year vs. 2 year) (mean ± SEM, n = 4–10 mice/group, One-way ANOVA).