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

Figures

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  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…

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  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).

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  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).