Cerebral Focal Stroke Causes Increased Incidence of Non-Flowing Capillaries Hundreds of Micrometers into the Adjacent Microvasculature

Introduction: Focal ischemic strokes are small enough that they do not lead to acute clinical symptoms, but they are, nonetheless, closely associated with cognitive decline. In mouse models of such focal strokes, an increased incidence of non-flowing capillary segments in the adjacent tissue has been observed. Administration of antibodies against the neutrophil-specific cell surface protein Ly6G leads to a decrease in the number of these stalled capillaries, resulting in increased penumbral blood flow, decreased infarct size, and improved behavioral outcomes, suggesting that improving microcirculatory blood flow is a promising therapeutic target for stroke. While previous work has shown functional improvements after decreasing stalls, the spatial extent and temporal dynamics of stalling after a focal stroke remain unknown. Here, we evaluate the dynamic capillary stalling response in vivo using a focal stroke model and characterize the cell types involved in capillary stalls.

Materials and

Methods: Photothrombotic focal stroke model: Mice with green fluorescent protein (GFP) knocked in for the Cx3cr1 gene were used in this study, labeling microglia. Chronic cranial windows were implanted on 3-month old mice (n=2 female, n=1 male). Four weeks after the craniotomies, photothrombosis was performed to create a focal stroke in single cortical penetrating arterioles. Rose Bengal dye (10 mg/mL in saline) was retro-orbitally injected immediately followed by excitation with a green laser (0.48 mW) for 30-60 seconds focused on the target vessel ~100 µm beneath the surface to induce occlusion. In vivo imaging: Two-photon excited fluorescence microscopy was used to identify stalled capillaries and characterize the cells present in the stalled vessels. The microvasculature was fluorescently labeled with retro-orbitally injected Texas-red dextran (40 µL, 2.5%, MW = 70 kDa). Rhodamine-6G (1 mg/mL, 100 µL) and Hoechst (4.8 mg/mL, 50 µL) were also injected to differentially label cellular components of the blood (Table 1). Z-stacks were taken up to 500 µm deep in three locations starting centered over the occluded vessel and including two adjacent ROIs (Fig. 2A). Stacks were collected at baseline and immediately after photothrombosis. Stall analysis: Stalls were identified as vessels with no flow for 5 s or longer (Fig. 1A), and cells involved in the stall were characterized by their fluorescent signature (Table 1, Fig. 1B). The number of stalled capillaries per unit volume was quantified for each z-stack. Two-way ANVOAs were performed to compare the number of stalls and cell types involved in the stalling events.

Results, Conclusions, and Discussions: There was no difference in the number of stalls at any distance from the targeted stoke vessel at baseline (p>0.26, Fig. 2A, B). After occlusion, there was a significant increase in the number of capillary stalls up to 0.5 mm away from the targeted stroke vessel (Fig. 2B), and a trend toward increased stalls up to ~1 mm away (p=0.18). These data show that targeting a single penetrating arteriole for occlusion had immediate effects on capillary blood flow as far as 0.5 mm away from the occluded vessel. Additionally, these blood flow changes occurred far outside the area where Rose Bengal was optically excited, indicating these increases in stalls in the adjacent microvasculature were not a result of direct Rose Bengal dye excitation in these capillaries, and rather are a reaction to the occlusion of a penetrating arteriole. From the three fluorescent markers, the cell types in each stalled vessel were characterized at baseline and after stroke. There was a significant increase in the number of stalls containing RBCs and platelets after stroke, as compared to baseline (p < 0.001 and p< 0.01, respectively; Fig. 2C). Interestingly, there was no change in the number of stalls containing leukocytes at baseline compared to immediately after stroke (p=0.44, Fig. 2C). Future work will evaluate the cell types involved in capillary stalls up to 14 days after stroke.
Overall, these data show that occluding a single penetrating arteriole has immediate effects on capillary blood flow as far as 0.5 mm away from the occluded vessel. Understanding the dynamics of cell types causing capillary stalls will allow for the investigation of cellular mechanisms driving these cell arrests and guide the development of targeted therapies to improve blood flow in the brain tissue surrounding a focal stroke. Notably, ischemic stroke drives increased inflammation, and microglia have been implicated in the inflammatory response to cerebrovascular injury. The mouse strain used in these experiments have GFP-tagged microglia, and current work includes characterization of the microglia response to the focal stroke injury to further understand the role these cells play in capillary degeneration.

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