Local hemodynamics dictate long-term dendritic plasticity in peri-infarct cortex

Abstract

Changes in dendritic spine turnover are a major mechanism of experience-dependent plasticity in the adult neocortex. Dendritic spine plasticity may also contribute to functional recovery after stroke, but in that setting its expression may be complicated by alterations in local tissue perfusion, especially around the infarct. Using adult Thy-1 GFP-M mice, we simultaneously recorded long-term spine dynamics in apical dendrites from layer 5 pyramidal cells and blood flow from surrounding capillaries with in vivo two-photon microscopy in peri-infarct cortex before and after unilateral middle cerebral artery occlusion. Blood flow in peri-infarct cortex decreased significantly immediately after stroke and improved gradually over time, in a distance-dependent manner from the epicenter of the infarct. However, local tissue perfusion was never fully restored even after a 3 month recovery period. On average, surviving layer 5 pyramidal neurons experienced a ∼20% decrease in spine density acutely after stroke but eventually recovered. The dynamics of this improvement were different depending on the degree of tissue perfusion acutely after arterial occlusion. Cells in ischemic areas closer to the infarct returned to normal spine density levels slowly by retaining spines, while cells in more remote regions with preserved blood flow recovered faster by adding more spines, eventually surpassing baseline spine density by 15%. Our data suggest that maintaining tissue perfusion in the area surrounding the infarct could hasten or augment synaptic plasticity and functional recovery after stroke.

Figures

Figure 1.

Unilateral permanent MCAO model of stroke and experimental design. a, Location of cranial window (orange circle) and site of MCAO in relation to the structure of the adult mouse skull [left, three-dimensional side view (Image courtesy of DigiMorph.org); right, top view]. b, Location of the cranial window in relationship to the MCA vascular territory (red). c, Location of the cranial window in relationship to functional brain regions (loosely based on data from Diamond et al., 1999; Paxinos and Franklin, 2001; Grove and Fukuchi-Shimogori, 2003; Van der Gucht et al., 2007). Green shading is for primary somatosensory cortex (BF, barrel field; FL, forelimb; HL, hindlimb; LL, lower lip; No, nostril; Rv, rostral vibrissae; Tr, trunk; UL, upper lip), blue shading is for visual cortex and red shading for motor cortex. d, Thirteen coronal sections from a mouse in this study, showing the location and size of a representative infarct after MCAO (the red shaded area represents the portions of cortex that was missing when the mouse was perfused at +94 d). e, Low-magnification bright field image of a coronal section of the same GFP-M mouse. f, Fluorescent photomicrographs of the same coronal section at increasing magnification (from left to right) showing the area that was chronically imaged with two-photon microscopy. g, Appearance of blood vessels as seen under the cranial window (dashed circle) in a typical experiment. The typical appearance of neuronal structure in zones 1 and 2 before and after stroke are shown in higher magnification in i. R, Rostral; C, caudal; L, lateral; M, medial. h, Diagram of a virtual cranial window showing the location of all the dendrites imaged in this study (each circle depicts a region of interest). The shaded area in the rostral-lateral corner depicts a region in which neuronal structure was reliably damaged after stroke. i, Side projections (xz) of image stacks (range 65–75 slices, 5 μm apart) in zone 1 (top) and zone 2 (bottom) before the stroke. j, k, Maximum intensity projections of the same image stacks before (j) and after (k) the stroke. In general, dendrites located closest to the infarct (area 1) were destroyed. Some were still identifiable and could be used as reference landmarks (yellow arrowheads). We avoided imaging dendrites under blood vessels (red dashed line) because the GFP signal was obscured. l, Experimental design for time lapse two-photon imaging of dendrites and blood flow.

Figure 2.

Chronic imaging of dendritic structure in peri-infarct cortex before and up to 3 months after MCAO. a, High-resolution two-photon microscopy images acquired in vivo of an apical dendritic segment from an L5 pyramidal neuron in peri-infarct cortex. All are maximum intensity projections (4–7 slices, 1.5 μm apart). A few examples of always-present spines (yellow arrows), gained spines (green arrowheads), and lost spines (open red arrowheads) are shown. The day of imaging is shown in the upper left corner. Blue asterisks at +4 and +8 d post-MCAO denote transient dendritic swelling after stroke. b, Normalized spine density over time for 26 L5 pyramidal neurons from 6 mice. c, Normalized spine density averaged across neurons in stroke mice (red) and control mice (black; 22 neurons from 5 mice). Data are shown as means ± SEM (two-way ANOVA followed by Bonferroni's multiple-comparisons test: **p < 0.01; ***p < 0.001). The vertical dashed line in b and c indicates the time of MCAO.

Figure 3.

Altered spine dynamics underlying the recovery of dendritic spines. a, Number of gained and lost spines per unit length of dendrite as a function of time, before and after MCAO. The vertical dashed line indicates the time of MCAO. Dashed lines (blue and green) indicate the baseline values for lost and gained dendritic spines before MCAO. b, Ratio of the density of lost/gained spines in control mice (black; average of all time points) and in stroke animals (red) as a function of time. Gray shading indicates the baseline ratio before MCAO. c, Half-life of spines in control and stroke animals at various intervals after MCAO. Error bars represent the SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 10−4).

Figure 4.

Dramatic and long-lasting reductions in blood flow in peri-infarct cortex. a, Low-magnification view of the blood vessels on the cortical surface as seen through the glass-covered cranial window. b, Higher-magnification view of the boxed region in a. c, Rhodamine B angiogram of the cortical vasculature (in red) corresponding to the same region shown in b. GFP-expressing dendrites of L5 pyramidal neurons can also be seen (green). This is a maximum intensity projection of a stack of images (75 slices, 5 μm apart) obtained with two-photon microscopy after systemic injection of rhodamine B-dextran through the tail vein. d, Higher-magnification view of the boxed region in c, showing dendrites in green and a capillary in red. e, Line scan (xt) along the yellow line through the capillary shown in d. Each oblique black line represents a single RBC that displaces the fluorescent dye as it travels through the capillary from left to right. From these graphs we could calculate RBC velocity as Δx/Δt). f, Representative xt line scans through a capillary in a control animal (left) at various times, and a capillary in a stroke animal (right) before (PRE) and at various intervals after MCAO. g, Scatter plot of RBC velocity and flux in stroke animals before (PRE), at 1 h after, and at 90 d after MCAO (n = 74, 59, and 45 capillaries, respectively, from 3 mice). Pearson's r = 0.79, 0.88 and 0.78, respectively (p < 0.001 for all datasets) between flux (RBCs/s) and velocity (mm/s). Linear densities ρ−1 = 11.8 ± 0.5, 10.3 ± 0.5, and 10.8 ± 0.6 μm, respectively (n.s. between the different time points). h, Normalized RBC flux for control (black) and stroke (red and blue) animals as a function of time before and after MCAO. Red trace shows data from capillaries surrounding dendritic segments from cells that were present during the whole length of the study. The blue trace also includes data from capillaries surrounding dendritic segments from cells that disappeared after the MCAO. The vertical dashed line indicates the time of MCAO. Error bars represent the SEM (Bonferroni's multiple-comparisons test: **p < 0.01; ***p < 0.001). i, j, Normalized spine density for stroke animals as a function of RBC flux at +4 d after stroke (i) and for +20, +24, and +28 d post-MCAO (j) (Pearson's: *p < 0.05).

Figure 5.

Acute local blood flow after stroke predicts the mode of spine recovery. a, Frequency distribution of normalized RBC flux in control mice at 1 h time point (black) and in stroke mice at 1 h after MCAO (red). The upper 95% confidence interval for the distribution in stroke animals (∼0.7 of normalized RBC flux) is indicated by the vertical dashed line. In stroke animals, cells that exhibited average capillary RBC flux below this value (rounded to 0.7 of normal) were grouped together as regions with blood flow (BF) <70% and those above this value were grouped as BF>70%. b, Normalized spine density as a function of time before and after MCAO for cells exposed to ischemia (BF<70%, red) or to intact tissue perfusion (BF>70%, green). For each dendritic ROI, the RBC flux values were averaged for 1–4 capillaries (average 2.7 per dendrite) in its immediate vicinity. Next, cells were assigned to either BF category based on the average RBC flux for all its dendritic ROIs. Solid circles indicate statistically significant differences from baseline (pre-MCAO), and the asterisk (p < 0.05) indicates a statistically significant difference between the two BF groups (two-way ANOVA followed by Bonferroni's multiple-comparisons test). c, Number of gained spines per unit length of dendrite at various times before (PRE) and after MCAO for both BF groups (two-way ANOVA followed by Bonferroni's multiple-comparisons test; *p < 0.05). d, Number of transient spines per unit length of dendrite before (PRE) and at +4 and +24 d after MCAO for the two BF groups (two-way ANOVA followed by Bonferroni's multiple-comparisons test; *p < 0.05). e, Half-life of spines in the two BF groups at various intervals after MCAO (extra sum-of-squares F test; *p < 0.05; **p < 0.01). f, Example of new persistent spines (arrows) emerging at +60 d on a dendrite with reduced blood flow and lasting until +90 d after MCAO. g, Fraction of new persistent spines appearing at various times after MCAO for the two BF groups (two-way ANOVA followed by Bonferroni's multiple-comparisons test; *p < 0.05). Gray shading in d, f, and h shows the values in control mice without stroke. Error bars represent the SEM.

Figure 6.

The magnitudes of spine recovery and local tissue perfusion correlate with distance away from infarct. In all four panels, the cranial window is shown as a blue circle. The branching pattern of a representative MCA as seen through the window is shown in white as an example. The white cloud represents the maximum intensity projection of all MCA branches in all animals imaged (controls and stroke). The generic cranial window is divided into three concentric regions, A–C, each 1 mm farther away relative to the infarct core. a, b, Normalized spine density at +4 d (a) and +90 d (b). Each square represents a different cell (n = 26 cells, from 6 mice). When several cells overlapped in the same region, separate neighboring boxes were assigned to different cells. The color of each box reflects the normalized spine density for a given cell, according to the color map on the right. Insets depict the average spine density values ± SEM for regions B and C. Unpaired t test: *p < 0.05. c, RBC flux at 1 h after MCAO obtained by rhodamine B-dextran angiography. Each square represents the average normalized RBC flux for a given cell (n = 258 capillaries at +1 h). When data for two or more cells overlapped in the same region, separate neighboring boxes were assigned to different cells. The color of each box reflects the normalized RBC flux in that cell, according to the color map on the right. Inset depicts the average RBC flux values ± SEM for regions A–C. One-way ANOVA followed by Bonferroni's comparison test: *p < 0.05. d, Relative cerebral blood flow at +1 h after MCAO obtained by laser speckle contrast imaging (values are relative to baseline using the same pseudocolor scale as in c). The image represents the average relative BF throughout the field of view in the cranial window obtained after transforming speckle contrast values (K) to cerebral blood flow values (see Materials and Methods for details). Data from 4 different mice were superimposed and averaged onto a single image. Inset depicts the average relative BF values ± SEM for regions A–C. One-way ANOVA followed by Bonferroni's comparison test: **p < 0.01. R, Rostral; C, caudal; L, lateral; M, medial. These maps show that spine density reached levels above baseline for region C, which is farthest away from the infarct and also the one with more preserved blood flow at +1 h. c has more boxes than a because we could measure blood blow at +1 h in more regions than we could follow dendritic structure eventually at +4 d.