Figure S1: Rapid Clearance of RB after Intravenous Injection
RB was injected into the tail vein of a YFP-H mouse and two-photon imaging of both red and green fluorescence was started about 2 min later.
(A) Relatively weak RB fluorescence was observed with two-photon excitation ~2 min after injection. Most of the RB signal was gone within 10 min (Tau = 175 ± 29 s).
(B) Time course of RB fluorescence detected by two-photon microscopy using a red emission channel, single 1-μm section shown. In this graph changes in red fluorescence (minus any preinjection offset) divided by YFP fluorescence (to control for small time dependent changes in imaging conditions) are plotted for 6 h. During the first 10 min after injection (when RB levels were highest) only single scan images (1-μm sections) were taken to reduce the chance on-going photoactivation. After this time the brain was scanned repeatedly (at least eight times) at high resolution by taking 100, 1-μm sections as was typically done for timelapse imaging.
(C) High power image of spiny dendrites (a single 1-μm section is shown) before and 6 h after RB injection shows that without photoactivation of RB by green light, little dendritic damage occurs (similar results were obtained in a total of five animals).
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Figure S2: Laser-Induced Photoactivation Does Not Lead to Direct Dendritic Damage
(A) Video image showing surface vasculature illuminated by a green LED.
(B) A higher-magnification view of the boxed region in (A) showing both dendritic and vascular structure (merged GFP and TR-dextran image). A RB-containing surface arteriole was photoactivated at several points (green arrows) near where it penetrated the brain. A nearby fine dendritic branch was within 15 μm of the photoactivation area, but showed little acute damage after over 15 min of alternating photoactivation with a focused laser at the three sites. Clots can be seen developing at the three sites. The preservation of dendritic structure was attributed to only partial blockade of blood flow supplying the area of the branch. For creation of the color merged version (only) nondendritic green channel material was manually masked.
(C) Dendritic structure before and 15 min after local RB laser activation.
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Figure S3: Effect of MCAO on Dendritic Structure
(A) Ligation of the right CCA unilaterally did not cause dendritic damage within ~5 h within the right somatosensory cortex (necessary to perform MCAO). Red, blood vessels; green, dendrites.
(B) TR-dextran assessment of blood flow indicated a significant reduction in flux and velocity after unilateral ligation of the CCA. The flux for pretreatment was 108 ± 10 RBCs per second, and the velocity for pretreatment was 1,340 ± 230 μm/s.
(C) In another animal unilateral ligation of the CCA followed by suture occlusion of the MCAO lead to rapid and extensive dendritic damage even with significant residual blood flow. Flowing vessels can be assessed by the streaking caused by scanning of moving RBCs. The yellow arrowhead shows a clotted capillary, and the blue arrowhead shows a flowing capillary at 30 min. All vessels are clotted at 105 min in the region shown.
(D) Average blood flow change from three monitored capillaries in the animal after MCAO. The flux for pretreatment was 123 ± 12 RBCs per second, and the velocity for pretreatment was 1,290 ± 80 μm/s.
(E) Area of clotted vessels (expressed as percentage of prestroke total vessels) increased, and spine number decreased with time after MCAO. Within the MCAO model, the percentage of flowing vessels, the RBC flux, and RBC velocity were reduced.
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Figure S4: Dendritic-Damage Rating Scale
To qualitatively assess dendritic damage after RB photothrombosis, we used a five-point rating scale: 0, no damage; 1, blebbed dendrites < 5%; 2, 5%–25% blebbed dendrites; 3, 25%–50% blebbed dendrites; 4, 50%–75% blebbed dendrites; 5, 75%–100% blebbed dendrites. Arrowheads show some examples of blebbed dendrites.
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Figure S5: Large Flowing Vessels at the Border of Dendritic Damage
(A) Maximal intensity projection of TR-dextran labeled vessels 100 min after photothrombotic stroke induction using a fluorescence arc lamp. In this example TR-dextran was injected after photothrombosis to reduce the incidence of extravasation. Flowing small vessels were colored red. A single large flowing vessel, likely a vein (dashed line), formed the border between relatively intact dendritic arbors on the left and severely damaged structure on the right.
(B) Dendritic damage borders: a 50-μm maximal intensity projection of dendrites is shown. A total of five different dendrite-damage border regions produced at 10-μm Z-intervals are shown in green. We then made measurements of distance between these borders and the nearest flowing small (<15 μm) or large vessel. The blue boxes in (B) indicate areas in which dendritic damage borders were examined. Because it was possible that flowing vessels could be located near the edge of an image, a 50-μm buffer zone indicated by the blue line was used to avoid measurements near the border.
(C) Example of a banded vessel indicated in (A) showing blood flow. A total of three different 1-μm Z-sections are shown through this vessel, and variation in banding pattern in different sections is indicative of robust blood flow.
(D) Example of a stalled vessel in which very little flow and trapped cells are apparent. No streaking or banding pattern was observed.
(E) Laser-speckle blood flow image showing the distribution of ischemia 15 min after photothrombotic stroke induction (dark areas indicate low contrast and blood flow, images scaled from 0%–10% contrast). The area imaged at high power using two-photon microscopy is indicated and is shown to be at the edge of a large ischemic region. Flowing venous branches are observed coming off the large vein. After the dendritic damage border region was found to deteriorate 205 min later, we performed another round of speckle imaging and found even more extensive clotting and that the blood flow border had moved considerably.
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Figure S6: Loss of Functional Maps and EcoG after Severe RB Stroke
(A) Video image showing surface vasculature illuminated by a green LED before and after RB stroke. A Teflon-coated silver wire (arrowhead) was placed on the surface of the cortex within the agarose to record an EcoG.
(B) Severe ischemia (2.2 mm2 of cortical surface area affected) completely abolished the contralateral hindlimb movement-evoked functional map (darkened area, expressed as a percentage change in the 635-nm reflected light signal averaged over 40 stimulus trials taken at least 20 s apart).
(C) After severe stroke the EcoG was also markedly suppressed.
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Figure S7: Cortical Hemodynamic Responses within 200 μm of Damaged Dendrites after RB-Induced Photothrombosis
(A) Contralateral hindlimb stimulus evoked IOS map before and after RB photothrombosis. The map was thresholded and overlaid on an image of the vasculature image.
(B) A two-photon image shows dendritic structure after RB stroke in the blue-boxed region in (A). Yellow line marks the same position in (A) and the dendritic damage border.
(C) Intensity profile across the functional map before and after RB stroke for the red-boxed region in (A). The red arrow indicates the half maximal intensity for the poststroke plot indicating that some cortical function is retained in the structurally intact tissue several hundred μm from the stroke edge. The blue arrow shows the location of the structural edge of the stroke and is also marked by the yellow line in (A) and (B).
(D) Raw IOS signal change in the hindlimb area before and after stroke scaled between −0.1 and 0.1%.
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