John Nguyen, Nozomi Nishimura, Robert Fetcho, Costantino Iadecola, Chris B. Schaffer

Transcription

1 Supplemental figures and text for Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries John Nguyen, Nozomi Nishimura, Robert Fetcho, Costantino Iadecola, Chris B. Schaffer Supplemental Figure 1 Histogram of baseline diameter and speed for all measured capillaries and venules. For this study, 298 capillaries were measured with a baseline median (A) diameter and (B) speed of 5.4 µm and 0.49 mm/s, respectively. Additionally, 102 surface venules were measured, with a baseline (C) diameter and (D) speed of 33 µm and 0.61 mm/s, respectively.

2 Supplemental Figure 2 Histogram of baseline diameter and speed of occluded venules. Sixteen AVs were targeted with a baseline median (A) diameter of 18.4!m and (B) speed of 0.32 mm/s. Eight SVs were targeted with a baseline median (C) diameter of 39.3!m and (D) speed of 0.59 mm/s.

3 Supplemental Figure 3 Ascending venule occlusion case study one. Average projection of a 2PEF image stack before (A) and after the clot (D), with the targeted venule indicated by the blue circle in A. Baseline (B) RBC velocities and (C) diameters are shown in upstream capillaries, with post-clot measurements in (E) and (F), respectively. Arrows indicate the direction of blood flow. Double arrowheads and bold values indicate vessels that reversed in flow direction following the occlusion and the red X indicates the location of the clot.

4 Supplemental Figure 4 Ascending venule occlusion case study two. Same panel layout as Supplemental Figure 3.

5 Supplemental Figure 5 Surface venule occlusion study with a collateral vessel present. Same panel layout as Supplemental Figure 3. The collateral vessel (purple) connects the terminal vessel to another surface venule (dashed line). Note that in this example, where a collateral vessel is present, the capillaries upstream from AVs (blue circles in A) did not reverse in flow direction.

6 Supplemental Figure 6 Surface venule occlusion study with no collateral vessel present. Same panel layout as Supplemental Figure 3. Note that with no collateral vessel present, some capillaries upstream of AVs reverse in flow direction following the occlusion.

7 Supplemental Figure 7 Boxplot of the nearest-neighbor distance between two AVs and two PAs. The average distance between two PAs (145.9 µm ± 5.2 µm (mean ± SEM)) is significantly greater (p < 0.01, t-test) than the average distance between two AVs (123.6 µm ± 2.6 µm).

8 Supplemental Movie 1 Occlusion of an ascending venule. This video shows the occlusion of a single cortical venule in an anesthetized rat. The fluorescently-labeled surface vasculature is imaged at the beginning of the video. Note the red blood cells are unlabeled and appear as dark patches whose motion through the vessels indicates blood flow. The video then zooms in on the targeted segment of the AV, approximately 40 µm below the brain surface, and the procedure for inducing the clot commences. The vessel lumen is targeted with high-energy femtosecond laser pulses, represented by white flashes streaking across the frame. Once the vessel is sufficiently injured, extravasation of plasma and RBCs is observed and irradiation of the vessel continues until the injury is sufficient enough to initiate clot formation. To determine whether or not a clot has formed, we look for a cessation of RBC motion in the downstream surface segment of the venule at the brain surface. In this example, after the initial injury, we see that the blood flow is sluggish, but has not completely ceased. The AV is then irradiated again until the clot has fully formed and flow stops. This video is played at three times real time.

9 Supplemental Text. Here we provide a step-by-step walkthrough of the flow change analysis shown in Figure 6D. To start, Figure 6C shows the capillary branch number as a function distance from either an AV or a PA. For a given spatial window, we determine the number of vessels in that spatial region for each branch number. The data table above is an example for the region 0-50!m away from an AV. The first column lists the same capillary branch number groups as seen in Figure 4. The number of capillaries at each capillary branch number within this 50-!m window is shown in column two. Column three gives the average in vivo RBC speed following an AV occlusion for capillaries with different branch number as a fraction of the baseline value. For each branch number, the number of vessels counted is multiplied by the average post-clot speed to give a weighted speed change (column four). The weighted values are then summed to calculate the total weighted speed change for this spatial region. This value is then divided by the total number of vessels counted to give the average weighted post-clot flow for the 50-!m window. In this example, the average weighted flow for the first 50!m from an AV is 44% of baseline speeds. This procedure is then repeated for the next 50-!m window, and for both AVs and PAs to generate the data shown in Figure 6D.