Microvascular plasticity in mouse stroke model recovery: Anatomy statistics, dynamics measured by longitudinal in vivo two-photon angiography, network vectorization

Abstract

This manuscript quantitatively investigates remodeling dynamics of the cortical microvascular network (thousands of connected capillaries) following photothrombotic ischemia (cubic millimeter volume, imaged weekly) using a novel in vivo two-photon angiography and high throughput vascular vectorization method. The results suggest distinct temporal patterns of cerebrovascular plasticity, with acute remodeling peaking at one week post-stroke. The network architecture then gradually stabilizes, returning to a new steady state after four weeks. These findings align with previous literature on neuronal plasticity, highlighting the correlation between neuronal and neurovascular remodeling. Quantitative analysis of neurovascular networks using length- and strand-based statistical measures reveals intricate changes in network anatomy and topology. The distance and strand-length statistics show significant alterations, with a peak of plasticity observed at one week post-stroke, followed by a gradual return to baseline. The orientation statistic plasticity peaks at two weeks, gradually approaching the (conserved across subjects) stroke signature. The underlying mechanism of the vascular response (angiogenesis vs. tissue deformation), however, is yet unexplored. Overall, the combination of chronic two-photon angiography, vascular vectorization, reconstruction/visualization, and statistical analysis enables both qualitative and quantitative assessments of neurovascular remodeling dynamics, demonstrating a method for investigating cortical microvascular network disorders and the therapeutic modes of action thereof.

Keywords: Microvascular plasticity; in vivo two-photon angiography; longitudinal imaging; mouse stroke model; vascular network vectorization.

Figure 1.

Experimental Methods: (a) Mouse cortex is imaged in vivo through a cranial window using a tiling protocol to cover as large of a volume as possible in a single imaging session. The stitched image is vectorized using the SLAVV software for reconstruction, visualization, and statistical analysis. The vessel directions (as well as the borders of the 2PM images and the ROI borders) are color-coded with respect to their alignment with the imaging coordinate system: (xyz<->CMY). (b) LSCI is used to orient the 2PM imaging session to reproducibly image the same (∼1 mm³) volume longitudinally over several imaging sessions at two-week intervals and at a depth greater than 600 micrometers. Displayed is an orthographic projection in the (optical) z-axis of a tiled image volume of a healthy control subject. Lateral orthographic projections show the longitudinal imaging reproducibility and (c) the longitudinal experiment is repeated around a photothrombotic injury. The infarct appears to be contained to a (yellow) circle with a radius of approximately 1 mm in the LSCI post-stroke image. The x-projected 2PM image reveals that a (transparent cyan) spherical ROI beneath the surface approximates the shape of the infarct. Scale bars are 200 μm.

Figure 2.

3D Directional Analysis: (a) A 3D perspective rendering shows the entire capillary network captured in the rectangular 2 × 3 tiled image. The 1 mm radius spherical ROI (500 micrometers line also shown) intersecting the near image boundary is used in the stroke model analysis. The pop-out magnifies an example penetrating (blue) vessel and surrounding vessel strands inside of a (250 micrometer diameter, 600 micrometer height) cylindrical ROI. (b) The strands from the cylindrical ROI overlay MIPs of a rectangular volume ROI of the raw 2PM image in x-, y-, and z-orthographic projections. (c) All vessel sections in the 3D reconstructions and those belonging to strands in the cylindrical ROI in the 2D projections (as well as the ROI borders) are color-coded according to their alignment to the axes of the imaging system (xyz<->RGB). (d) The 1 mm radius spherical ROI is magnified and re-colored according to its orientation to the ROI center and (e) visual legend describing the concepts of vessel strands vs. sections and the distance and orientation statistics with respect to the spherical ROI center.

Figure 3.

Blood flow following ischemia as determined from MESI: (a) ICT maps determined from MESI at each time point for one mouse. The day zero image was acquired immediately before photothrombosis, all others are labeled with the day after photothrombosis. (b) Regions where blood flow was determined for one mouse. The regions indicated in shades of red are for the ischemic area, the region indicated by blue is for the reference region. This image is the same as that shown on the far right in part A, and images in A are shown with the same display range indicated by the colorbar in B and (c) plots of average relative blood flow with time. Blood flow for the region closest to the infarct is shown in the darkest shade of red, flows for regions further away are shown in lighter shades. Blood flow for the reference region is shown by the dashed blue line. Error bars represent standard error.

Figure 4.

Pre- and Post-Stroke Snapshots by Subject: To compare the variation between stroke model subjects, week 0 pre- and week 4 post-stroke time points from three mouse subjects (A, C, and E) were imaged, vectorized, and analyzed. Similar spherical ROIs were selected between subjects, concentric with the infarct center observed at week 4. Vessels appear to be removed from or reoriented and pulled toward the infarct center to different degrees between subjects.

Figure 5.

Longitudinal Stroke Model Snapshots: (a) Orthographic lateral MIPs of a rectangular volume (200 micron scale bar) of a raw 2PM image (concentric with the spherical ROI in the projection coordinate) for weeks 0, 1, and 4 post-photothrombosis. The orientation coloring for all vessel strands inside the (1 mm spherical) ROI overlays the original MIP. The tissue appears to contract toward the infarct center. (b) 3D perspective rendering of the strands within the ROI. A smaller (0.5 mm) ROI representing the core of the infarct is highlighted in red in week 0. Vessels in the core appear to be eliminated by the photothrombosis by week 1 and partially regrown by week 4 and (c) the time point renderings of the healthy control mouse B are shown for comparison.

Figure 6.

Longitudinal Stroke Model Statistical Analysis: The legend shows the color scheme given to the different snapshots (all mice have the same color at each time point). The healthy snapshots are darker colors, while the snapshots after photothrombosis begin red and fade to grey. The different marker symbols encode different comparisons (either vs. previous or vs. baseline). (a) Distribution snapshots of the length-weighted statistics for all time points healthy or stroke model vascular response to photothrombosis (From Top): cumulative length distributions of distance and orientation with respect to the ROI center, length-weighted Continued.histograms, normalized to PDF, integrated to CDF, and differences of CDFs between different pairs of time points. The K-S Test Statistics (extreme values of the differences of CDFs) summarize the differences between pairs of distributions. (b) Time courses of several statistics of interest (From Top): Total vascular length inside the ROI, overall isotropy, distance and orientation box and whisker plots (0,25,50 (median), 75,100th percentile), and K-S Test Statistics of the Distance and Orientation distributions (vs. the previous or the baseline).