Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion - PubMed (original) (raw)

Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion

Chris B Schaffer et al. PLoS Biol. 2006 Feb.

Abstract

A highly interconnected network of arterioles overlies mammalian cortex to route blood to the cortical mantle. Here we test if this angioarchitecture can ensure that the supply of blood is redistributed after vascular occlusion. We use rodent parietal cortex as a model system and image the flow of red blood cells in individual microvessels. Changes in flow are quantified in response to photothrombotic occlusions to individual pial arterioles as well as to physical occlusions of the middle cerebral artery (MCA), the primary source of blood to this network. We observe that perfusion is rapidly reestablished at the first branch downstream from a photothrombotic occlusion through a reversal in flow in one vessel. More distal downstream arterioles also show reversals in flow. Further, occlusion of the MCA leads to reversals in flow through approximately half of the downstream but distant arterioles. Thus the cortical arteriolar network supports collateral flow that may mitigate the effects of vessel obstruction, as may occur secondary to neurovascular pathology.

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Figures

Figure 1

Figure 1. TPLSM of Fluorescently Labeled Cortical Vasculature In Vivo

(A) Low-magnification TPLSM image of fluorescently labeled brain vasculature in rat parietal cortex. The axes indicate the rostral (R) and medial (M) directions. In the inset is an image of latex-filled brain vasculature taken from Scremin [1], with a box that indicates the approximate size and location of a typical craniotomy and an arrow that identifies the MCA. (B) Tracing of the surface arterial vascular network from the image in (A). Branches of the MCA are indicated, as are representative examples of the communicating arterioles (CA) that form the surface network and diving arterioles (DA) that supply cortex. (C) Maximal projection of a TPLSM image stack through a cortical arteriole. The dark line indicates the location where the line-scan data were taken, and the arrow represents the direction of flow obtained from these scans. (D) Line-scan data from the vessel in (C) to quantify the flow of RBCs. Each scan is displayed below the previous one, forming a space–time image with time increasing from top to bottom of the image. The dark streaks running from upper right to lower left are formed by the motion of the non-fluorescent RBCs. The RBC speed is given by the inverse of the slope of these streaks; the direction of flow is discerned from the sign of the slope. (E) RBC speed along the center of the arteriole shown in (C) and (D) as a function of time. The periodic modulation of the RBC speed occurs at the approximately 6-Hz heart rate. The dotted line represents the temporal average of the speed. (F) RBC speed in an arteriole, averaged over 40 s, as a function of the transverse position in the vessel along horizontal (y) and vertical (z) directions. The parabolic curve represents the laminar flow profile that most closely matches the data, i.e., s = _A_·(1 − r/R)2 where s is the speed of the RBCs, r is a radius from the origin and corresponds to either the y or z direction, R is the measured vessel radius of 26 μm, and A is a free parameter (A = 10 mm/s).

Figure 2

Figure 2. Photothrombotic Clotting of Individual Targeted Surface Cortical Blood Vessels in Anesthetized Rat

(A) Maximal projection of TPLSM image stack showing several surface arterioles (red A) and venules (blue V). The green circle indicates the region of the targeted arteriole that will be irradiated with green laser light. The white box indicates the region and orientation of the images in (C). (B) Schematic illustration of the targeted photothrombotic occlusion of a vessel and experiment timeline. After baseline imaging and blood flow measurements, rose bengal is intravenously injected into the animal. Green laser light is focused onto the wall of the target vessel, which excites the rose bengal and ultimately triggers the natural clotting cascade. Surface vessels adjacent to the target vessel are not occluded because they are not exposed to the 532-nm irradiation. (C) Planar TPLSM images of photothrombotic clotting of a surface arteriole. The frame on the left is taken at baseline. The green circle indicates the region of the targeted arteriole that will be irradiated, whereas the white arrows indicate the blood flow direction, as determined from line-scan measurements in the targeted vessel and in the vessels downstream from the target. The numbers over the downstream vessels correspond to the numbered line-scan data shown in (D). The streaked appearance of the vessels is due to the motion of RBCs during the acquisition of the image. The center frame is taken after an intravenous injection of rose bengal and 2-min irradiation with 0.5 mW of 532-nm laser light. The vessel is partially occluded (indicated by green double arrow). The right frame is taken after one more minute of irradiation. The target vessel is completely clotted (indicated by red X) whereas surrounding vessels are unaffected. Stalled blood flow is indicated by the dark mass of clotted cells in the target region and the brightly fluorescent region of blood plasma upstream from the target region. Note that blood flow is maintained in the branches downstream from the target vessel by a reversal in the direction of blood flow in the center branch, as determined from the line-scan data in (D). (D) Baseline and post-clot line-scan data for the numbered vessels downstream from the target vessel shown in (C). The average RBC speed determined from the line-scan data is indicated for each case, with a positive speed taken to be along the baseline direction of flow.

Figure 3

Figure 3. Assay of Oxidative Stress and Vascular Disruption

Pimonidazole hydrochloride was introduced into the bloodstream at 1 h after targeted photothrombosis. At the end of this period, the animal was euthanized and transcardially perfused for brain tissue fixation. Labeling is illustrated for a closely spaced series of sections (within 150 μm). (A1–A4) The section, immunolabeled with an antibody against pimonidazole adducts (Hypoxyprobe), shows localization of adducts at uptake sites that are largely restricted to zones beneath the clot. Lateral and medial directions are labeled by M and L, respectively. The intermediate- and high-magnification views show immunolabeling across neural compartments, including parenchyma and blood vessels (A2 and A4). A mixed brightfield-fluorescent image shows that the fluorescein-dextran retained in the vessels and extravasated into the parenchyma overlaps the pimonidazole labeling (A3). (B1–B3) Immunolabeling of the platelet marker CD41 indicates sparse damage. A mixed image of fluorescein-dextran vascular retention and the CD41 immunoreactivity indicates that the damage is confined to just below the clot (B2). (C1–C3) Immunolabeling of tissue with the reactive astrocyte marker vimentin increased only marginally the labeling of vessels just below the clot (indicated by an asterisk). A mixed image of fluorescein-dextran retention and the vimentin immunoreactivity indicates that the damage is co-localized (C2).

Figure 4

Figure 4. Neuronal Integrity near Photothrombotic Clot

The animal was sacrificed 1 h after the disruption, and the tissue was harvested from below the target vessel shown in Figure 5C. (A) The section, immunolabeled with an antibody against MAP2, shows widespread staining of neurons. (B) Fluorescent imaging of propidium iodide counterstain. The stained endothelial cells demarcate vessels. Note the numerous nuclei with segmented lobes characteristic of aggregates of leucocytes within the vessel (clot). (C) High-magnification view shows subtle neuropathology in some cells, i.e., corkscrew dendrites (single arrows) and shrunken neurons with eccentric nuclei (double arrow).

Figure 5

Figure 5. Examples of Flow Changes that Result from Localized Occlusion of a Cortical Surface Arteriole

(A–C) On the left and right are TPLSM images taken at baseline and after photothrombotic clotting of an individual vessel, respectively. Left center and right center are diagrams of the surface vasculature with RBC speeds (in mm/s) and directions indicated. The red X indicates the location of the clot, and vessels whose flow direction has reversed are indicated with red arrows and labels. In the examples of panels (A) and (B) we show maximal projections of image stacks whereas the example in panel (C) shows single TPLSM planar images; the streaks evident in the vessels in these latter frames are due to RBC motion, and the dashed box in the diagrams represents the area shown in the images.

Figure 6

Figure 6. Compendium of Flow Changes following Localized Photothrombotic Clotting of Communicating Surface Arterioles

(A) Illustration of the four different classes of vessels considered, each delineated by their connectivity to the target vessel (indicated by an X). (B) Plots of post-clot RBC speed as a function of baseline RBC speed for each vessel class. The post-clot and baseline speeds were significantly correlated for the upstream and parallel vessels, but uncorrelated for the downstream vessels (Table 1). (C) Plots of post-clot vessel diameter as a function of baseline diameter. The pre- and post-clot diameters were correlated for all cases (Table 1). (D) Plots of post-clot volume blood flux as a function of the baseline value. The diagonal lines represent post-clot flux levels of 10% and 100% of baseline. As for the diameters, the pre- and post-clot fluxes were correlated for all cases (Table 1).

Figure 7

Figure 7. Quantitative Measurements of Flow Changes in Cortical Arterioles after Filament Occlusion of the MCA

(A) Example of flow changes observed following MCA occlusion. Left: projection of a TPLSM image stack taken at baseline. Center: tracing of the surface arteriole network from the image with the baseline blood flow speed and direction indicated in some vessels. Right: blood flow speed and direction during MCA occlusion. Red arrows and speed labels indicate vessels whose direction has reversed. The axes indicate the rostral (R) and medial (M) directions. (B–D) RBC speed, vessel diameter, and volume blood flux, respectively, during MCA occlusion as a function of baseline values. The baseline and occlusion values of the diameter and flux are significantly correlated (p < 0.005), with r2 = 0.92 and 0.26, respectively; the baseline and occlusion values of the speed are not significantly correlated.

Figure 8

Figure 8. Summary of Quantitative Measurements of Changes in Volume Blood Flux in Response to Single Microvessel Occlusion and MCA Occlusion

(A) Illustration showing topological relationship of vessels in (B) to (E) relative to the clotted arteriole (indicated with an X). (B–E) Histograms of the ratio of the post-clot flux to the baseline flux for vessels with different topological relationships to a photothrombotically clotted cortical surface arteriole: (B) upstream, (C) parallel, (D) first branch downstream, and (E) second to fourth branch downstream. (F) Histogram of the ratio of the flux measured during intra-luminal filament occlusion of the MCA to the baseline flux for cortical arterioles. In the interests of clarity, outliers with post-clot flux greater than twice the baseline flux were excluded from these histograms: one parallel vessel (ratio = 7), two D1 vessels (ratio = 2.5, 2.8), one D2 – 4 vessel (ratio = 11), and one filament occlusion vessel (ratio = 2.1). The arrows point to the mean values across all data points for each vessel class.

Comment in

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