Acute and chronic imaging of dendrite morphology for controls and after a deep microhemorrhage or ischemic lesion.

<p>Maximum intensity projections of 2PEF image stacks of Layer II/III cortical dendrites in YFP-H mice (<b>A</b>) in control regions, (<b>B</b>) after microhemorrhage of a single PA at 400 µm beneath the cortical surface (deep hemorrhage), and (<b>C</b>) after photothrombotic clotting of a single PA (RB clot). For the deep hemorrhage, the RBC-filled core is not visible because it was located deep in the cortex, hundreds of micrometers directly beneath the shallow dendrites imaged here.</p

Microhemorrhage size limited by clotting of vessel wall.

<p>(<b>A</b>) Median RBC core diameter as a function of time for mice receiving heparin infusion (green) and controls (red). Bold lines are running medians with 95% confidence intervals indicated by shaded areas. (<b>B</b>) RBC core diameter as a function of time for a PA that was irradiated twice (indicated by red pulses). (<b>C</b>) RBC core diameter measured at ∼90 s divided by RBC core diameter measured at ∼30 s for vessels irradiated either at 0 s (one irradiation) or at 0 s and ∼60 s (two irradiations). ***p<0.001; Mann Whitney U test.</p

Dynamics of RBC and blood plasma extravasation after laser-induced microhemorrhage.

<p>(<b>A</b>) Maximum intensity projection of a 2PEF image stack of cortical dendrites (green) and blood vessels (red), before and 1.5 hr after microhemorrhage. The spatial extent of the RBC core (blood plasma) is represented by a yellow (white) outline. (<b>B</b>) 2PEF imaging of bleeding dynamics after rupture of a single PA. A RBC-filled core (yellow outline) and diffuse plasma (white outline) expanded into the parenchyma after PA irradiation. (<b>C</b>) RBC core and (<b>D</b>) blood plasma diameter as a function of time after microhemorrhage.</p

Astrocyte activation and RBC breakdown products seven days after microhemorrhage.

<p>(<b>A</b>) Immunohistology for GFAP in coronally-sectioned tissue at lesion site and contralateral control region. (<b>B</b>) Bright-field image of coronally-sectioned tissue stained with cresyl violet (pink; neuronal cell bodies) and Prussian blue (black; RBC breakdown products).</p

Quantification of dendrite morphology after cortical microhemorrhage.

<p>(<b>A</b>) Fraction of identified dendrites that showed no signs of degeneration as a function of time for hemorrhages produced 20–100 µm beneath the cortical surface (shallow hemorrhage), 300–500 µm beneath the surface (deep hemorrhage), for photothrombotic occlusion of a PA (RB clot), and controls. (<b>B</b>) Dendrite exclusion diameter as a function of time after microhemorrhage (black). Red symbols indicate RBC core size. Error bars represent standard error of the mean (SEM). p-value compared to controls: * p<0.05, ** p<0.01, *** p<0.001, # p<0.0001, ## p<0.00001; Mann Whitney U test.</p

Acute and chronic imaging of microglia/macrophage response after microhemorrhage.

<p>(<b>A</b>) Maximum intensity projection of 2PEF image stacks of microglia/macrophages (green) and blood vessels (red) before and after microhemorrhage (left). Chronic dynamics of microglia/macrophage behavior after microhemorrhage (right). Inset at right shows reactive processes invading the RBC-filled core (indicated by yellow outline) at 1.5 hr after the lesion. (<b>B</b>) Microglia/macrophage density relative to baseline at different distances from the microhemorrhage as a function of time for homozygous and heterozygous CX₃CR1-GFP mice (p-value compared to control: * <0.05, †† <0.01; analysis of covariance). (<b>C</b>) Maximal distance from the center of the microhemorrhage to the furthest responsive microglia/macrophage as a function of time after the lesion. Red symbol indicates distance to furthest responsive cell after laser ablation in the cortical parenchyma with an energy similar to that used to induce a microhemorrhage. (<b>D</b>) Fraction of microglia/macrophages with processes directed toward the lesion after a microhemorrhage (p-value compared to control for homozygous (heterozygous) mice: * (†) p<0.05, ** (††) p<0.01, *** (†††) p<0.001, # (%) p<0.0001, ## (%%) p<0.00001; Mann Whitney U test). (<b>E</b>) Diameter of region where microglia/macrophages are excluded after microhemorrhage as a function of time. Average RBC core diameter for the heterozygous (homozygous) animals is indicated in green (red) at 0.5 and 1.5 hr post hemorrhage. Error bars represent the standard error of the mean (SEM).</p

Compression of brain tissue near a microhemorrhage.

<p>(<b>A</b>) Maximum intensity projections of 2PEF image stacks of cortical dendrites before and 1.5 hr after a microhemorrhage. (<b>B</b>) Tissue displacement map representing the magnitude and direction of dendrite displacement after the microhemorrhage shown in panel A. Manual (red) and automated (blue) measurements are both shown. (<b>C</b>) Radially-averaged dendrite displacement as a function of distance from the center of the microhemorrhage for the example in panels A and B. (<b>D</b>) Average dendrite displacement (black circles) and fit to Equation (10) (green line) as a function of normalized distance from the center of the microhemorrhage.</p

Microhemorrhages do not crush nearby capillaries, but blood flow speed is reduced.

<p>(<b>A</b>) Maximum intensity projections of 2PEF image stacks of blood vessels in the vicinity of the RBC core (yellow outline) before and 1.5 hr after a microhemorrhage. (<b>B</b>) Classification of capillary segments within 125 µm from the target PA, identified before the lesion, as flowing, stalled, or missing at 1.5 hr after the lesion. Error bars represent binomial 95% confidence intervals. (<b>C</b>) 2PEF images and space-time linescans of a capillary located ∼70 µm from the target PA before and 1.5 hr after a microhemorrhage. (<b>D</b>) Boxplot of capillary blood flow speed 1.5 hr after a microhemorrhage, expressed as a fraction of the baseline speed for capillaries within 125 µm from the target PA. In control measurements, no hemorrhage was produced. ***p<0.001; Mann Whitney U test.</p

Tracking of individual dendrites over 14 days after a microhemorrhage.

<p>(<b>A</b>) 2PEF image of dendrites (green) and blood vessels (red) at basel ine in GFP-M mice. The yellow X indicates the PA that was ruptured. (<b>B</b>) 2PEF imaging of dendrites located 170 µm from the target PA (yellow box in panel A) over two weeks. (<b>C</b>) 2PEF images from a control region.</p

Acute and chronic imaging of dendrite morphology after shallow microhemorrhage.

<p>Maximum intensity projections of 2PEF image stacks of Layer II/III cortical dendrites from YPF-H mice at different times after microhemorrhage of a single PA 30 µm beneath the cortical surface (shallow hemorrhage).</p