Article Figures & Data
Figures
- Figure 1.
A novel single-vessel mouse model of photothrombotic stroke. a, Representative scheme of the custom-made setup for photothrombosis occlusion of the distal branch of the MCA; for details, see Materials and Methods. b, Representative scheme of the main steps of photothrombotic occlusion of the distal branch of the MCA and corresponding images acquired during surgery. Left, Exposure of the MCA after craniotomy. Middle, Highlighting of the laser irradiation focused on the blood vessel. Right, Formation of the clot. Scale bar, 0.5 mm. c, Experimental timeline for the two groups MCAPT and EB. d, Representative brain slices labeled with NeuN antibody. To quantify the lesion volume, we analyze one slice every 300 μm. The image in the inset, acquired with a confocal microscope, shows a boundary region between the periinfarct cortex and the stroke core. Scale bar, 1.25 mm. e, Right Quantification (mean ± SEM) of stroke volume for the Sham group (0.1 ± 0.0001) and MCAPT group 1 week after photothrombosis (6.9 ± 0.1 mm3). *p = 2.29E-08 based on one-way ANOVA followed by a post hoc Tukey’s HSD test (n = 6). The error bar for the Sham group (n = 4) is below the minimum threshold. See also Extended Data Figure 1-1.
- Figure 2.
MCAPT induces severe dystonia in the acute phase after stroke: a, Representative pictures of mice during the clasping test. A score of 0 was assigned to mice with no clasping reflex; 1, 2, 3, and 4 were assigned respectively when one, two, three, and four limbs are retracted on the abdomen. b, Left, The clasping reflex revealed a tendency to higher clasping behavior after stroke both in the acute phase (at 1dpl) and at 1wpl. *p value based on one-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test: Pre-1dpl, p = 0; Pre-1wpl, p = 1.57E-05; 1dpl-1wpl, p = 0. Right, The wire-hanging test revealed a decrease in the strength of mice forelimbs 24 h after the damage. *p value based on one-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test: MCAPT Pre-1dpl, p = 1.83E-5; 1dpl Sham-MCAPT, p = 1.83E-5. c, The graph shows the weight of mice measured at the three time points. d, The graph shows the mortality rate 24 h and 1 week after the lesion (n = 15). See also Extended Data Figure 2-1.
- Figure 3.
MCAPT induces blood–brain barrier leakage and edema formation in the ipsilesional hemisphere. a, Dorsal (left) and lateral (right) pictures of a representative MCAPT brain of a mouse injected in the tail vein with Evans Blue dye right after photothrombosis. 1–8, Coronal sections of the same animal. Black arrows point to tissue swelling. b, Ex vivo quantification of the brain tissue presenting a blue signal in the Sham group and the EB group (1dpl) 1 d after the photothrombotic occlusion. *p = 0.003 based on one-way ANOVA followed by a post hoc Tukey’s HSD test (n = 6). c, The table shows the clasping test score and the extension of extravasation for each EB mouse. d, Brain water content evaluation 24 h after damage highlights the increase of wet weight in the ipsilesional hemisphere of MCAPT mice with respect to Sham mice. *p = 0.003 based on one-way ANOVA followed by a post hoc Tukey’s HSD test (n = 4). e, Tissue swelling evaluation 24 h after stroke shows the emergence of brain tissue distortion affecting the ipsilesional hemisphere of MCAPT mice. *p = 0.0001 based on one-way ANOVA followed by a post hoc Tukey’s HSD test (n = 4).
- Figure 4.
MCAPT increases astrocyte density in the peri-infarct area. a, A representative brain slice highlighted the IC and the 4 ROIs identified for the astrocytes analysis. Scale bar, 0.5 mm. b, Representative field of view of each ROI acquired with a confocal microscope. Scale bar, 45 μm. c, The graph shows the density (average ± SEM) of GFAP-positive cells in the 4 ROIs (IBZIL= 252.57 ± 33.07; RZIL = 104.63 ± 13.23; IBZCL = 133.64 ± 29.11; ICCL = 115.18 ± 26.894). p value based on one-way ANOVA followed by a post hoc Tukey’s HSD test. See Extended Data Table 4-1: IBZIL-RZIL, p = 0.00,721; IBZIL-IBZCL, p = 0.02,508; IBZIL-ICCL, p = 0.01278. See also Extended Data Figures 4-1 and 4-2.
- Figure 5.
MCAPT increases astrocyte complexity in the peri-infarct area of MCAPT mice. a, Representative image of an astrocyte analyzed with the Sholl method. b, The graph shows the distribution of the number of intersections for each radius in the 4 ROIs. p value based on two-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test. See Extended Data Tables 5-1, 5-2, and 5-3: radius 7: IBZIL-RZI, p = 2.51E-08; IBZIL-IBZCL, p = 0.006; IBZIL-ICCL, p = 0.006; radius 8: IBZIL-RZIL, p = 8.65E-07; IBZIL-IBZCL, p = 0.013; IBZIL-ICCL, p = 1.72E-04; radius 9: IBZIL-RZIL, p = 6.82E-06; IBZIL-IBZCL, p = 0.029; IBZIL-ICCL, p = 4.10E-04. c, Representative image of the same astrocyte in a analyzed with the Skeleton analysis. All the features of astrocytes in the 4 ROIs are shown as the average ± SEM. The intergroup statistical analysis was performed through a two-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test; see Extended Data Table 5-4. The intragroup statistical analysis was performed through a one-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test; see Extended Data Tables 5-5 and 5-6. Total branches length sham = 106.71 ± 3.32; IBZIL= 176.53 ± 15.04; RZIL= 128.32 ± 4.74; IBZCL = 152.23 ± 14.49; ICCL = 143.28 ± 11.04; intergroup analysis: IBZIL MCAPT-Sham, p = 0.002; intragroup analysis: IBZIL-RZIL, p = 0.003; IBZIL-ICCL, p = 0.033. Number of astrocytes, branches: Sham = 35.54 ± 1.68; IBZIL = 54.645 ± 3.126; RZIL = 43.127 ± 2.161; IBZCL = 47.307 ± 2.742; ICCL = 47.341 ± 2.113; intergroup analysis: IBZIL MCAPT-Sham, p = 0.001; intragroup analysis: IBZIL-RZIL, p = 0.044. Number of astrocytes, junctions: Sham = 17.18 ± 0.87; IBZIL = 26.81 ± 1.63; RZIL = 20.89 ± 1.13; IBZCL = 23.01 ± 1.421 ICCL = 23.04 ± 1.12; intergroup analysis: IBZIL MCAPT-Sham, p = 0.03; intragroup analysis: IBZIL-RZIL, p = 0.026; IBZIL-ICCL, p = 0.042. Number of astrocytes, end points: Sham = 18.68 ± 0.77; IBZIL = 27.47 ± 1.34; RZIL = 22.49 ± 0.86; IBZCL = 24.47 ± 1.20; ICCL = 24.58 ± 0.97; intergroup analysis: IBZIL MCAPT-Sham, p = 0.02; intragroup analysis: IBZIL-RZIL, p = 0.044. See also Extended Data Figures 5-1 and 5-2.
Extended Data
Figure 1-1
Sham mice ex vivo does not show sign of tissue suffering. On the right, panels 1–4 show representative coronal brain slices (100 μm thick) labeled with NeuN antibody 1 week after surgery. The ex vivo analysis does not find regions of tissue suffering or necrosis due to craniotomy or laser irradiation. Scale bar, 1 mm. On the left, panels 5–8 show representative coronal brain slices (1 mm thick), 24 h after surgery and intravenous injection of Evans Blue dye. The absence of blue staining highlights that the surgery followed by green laser illumination does not induce BBB permeability alterations. Download Figure 1-1, TIF file.
Figure 2-1
a, Body weight evaluation of Sham group at three different time points Pre, 1dpl, and 1wpl, respectively. b, As observed in MCAPT mice, the clasping reflex revealed a tendency to higher clasping behavior after stroke in the acute phase (1dpl) in the EB group as well as in the MCAPT group. *p value based on one-way repeated-measures ANOVA followed by post hoc Tukey’s correction: Pre-1dpl, p = 0.00002. c, Body weight monitoring does not highlight any alteration after the MCA occlusion. Download Figure 2-1, TIF file.
Figure 4-1
GFAP analysis. Representative images of GFAP-labeled astrocytes in the four different regions of interest (IBZIL, RZIL, IBZCL, ICCL) for each mouse. Download Figure 4-1, TIF file.
Figure 4-2
Astrocytes density in Sham mice: on the left, a representative image of anti-GFAP-labeled astrocytes. Scale bar, 45 μm. The graph on the right shows the density (average ± SEM) of GFAP-positive cells in the 4 ROIs (IBZIL = 18 ± 2.2; RZIL = 17 ± 3.6; IBZCL = 18 ± 3.1; ICCL = 20.7 ± 5.1). Download Figure 4-2, TIF file.
Table 4-1
Astrocytes density intragroup (Sham and MCAPT) and intergroups comparison. One-way repeated-measures ANOVA followed by Tukey’s test was used for intragroup comparison. Two-way repeated-measures ANOVA followed by Tukey’s test was used for intergroup comparison. Colored cells indicate p-values < 0.05. Download Table 4-1, DOC file.
Figure 5-1
Sholl analysis in MCAPT and Sham mice. The graphs show the distribution of the number of intersections for each radius in the 4 ROIs, color coded as in Figure 5. Download Figure 5-1, TIF file.
Figure 5-2
a, b, Skeleton analysis of astrocytes in MCAPT mice (a) and Sham (b) mice. All the parameters evaluated in the 4 ROIs are shown as the average ± SEM. The intergroup statistical analysis was performed through a two-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test (see Extended Data Table 5-4). The intragroup statistical analysis was performed through a one-way repeated-measures ANOVA followed by a post hoc Tukey’s HSD test (see Extended Data Tables 5-5, Tables 5-6). a, Junctions (pixel): Sham = 56.37 ± 3.37; IBZIL = 85.74 ± 4.93; RZIL = 68.45 ± 3.5; IBZCL = 74.01 ± 4.36 ICCL = 75.47 ± 4.2; intergroup analysis: IBZIL MCAPT-Sham, p = 0.00009. Average length of branches (μm): Sham = 4.28 ± 0.14; IBZIL = 4.35 ± 0.18; RZIL = 3.81 ± 0.24; IBZCL = 4.38 ± 0.18; ICCL = 3.96 ± 0.23; intergroup analysis: IBZIL MCAPT-Sham, p = 0.03; intragroup analysis: IBZIL-RZIL, p = 0.01; IBZCL-RZIL, p = 0.007; IBZCL-ICCL, p = 0.05. Maximum length of branches (μm): Sham = 14.01 ± 0.37; IBZIL = 16.11 ± 0.42; RZIL = 13.95 ± 1.04; IBZCL = 15.6 ± 1.37; ICCL = 13.53 ± 0.87. b, Total length of branches (μm): Sham, IBZIL = 112.914 ± 9.819; RZIL = 106.157 ± 5.648; IBZCL = 104.43 ± 6.303; ICCL = 193.352 ± 5.869. Number of astrocytes in branches (average ± SEM) in the 4 ROIs: IBZIL = 40.87 ± 5.15; RZIL = 34.49 ± 1.7 IBZCL = 34.05 ± 2.51; ICCL = 32.76 ± 2.84. Number of astrocytes in junctions: IBZIL = 19.91 ± 2.71; RZIL = 16.74 ± 0.87; IBZCL = 16.35 ± 1.29; ICCL = 15.73 ± 1.45. Number of astrocytes at end points in the 4 ROIs: IBZIL = 21.23 ± 2.28; RZIL = 17.79 ± 0.84; IBZCL = 18.19 ± 1.18; ICCL = 17.519 ± 1.317. Junctions (pixels): IBZIL = 67.39 ± 11.48; RZIL = 54.63 ± 2.02; IBZCL = 52.56 ± 3.55; ICCL = 50.88 ± 4.79. Average length of branches (μm): IBZIL = 3.9 ± 0.15; RZIL = 4.3 ± 0.25; IBZCL = 4.37 ± 0.21; ICCL = 4.54 ± 0.47. Maximum length of branches (μm): IBZIL = 13.90 ± 0.27; RZIL = 14.15 ± 0.62; IBZCL = 14 ± 0.51; ICCL = 14 ± 1.44. Download Figure 5-2, TIF file.
Table 5-1
Intragroup (MCAPT) comparison of Sholl analysis in different regions of the cortex. Two-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-1, DOC file.
Table 5-2
Intragroup (Sham) comparison of Sholl analysis in different regions of the cortex. Two-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-2, DOC file.
Table 5-3
Intergroup (MCAPT and Sham) comparison of Sholl analysis for each region of the cortex. Two-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-3, DOC file.
Table 5-4
Intergroup (MCAPT and Sham) comparison of Skeleton analysis for each region of the cortex. Two-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-4, DOC file.
Table 5-5
Intragroup (Sham) comparison of Skeleton analysis in different regions of the cortex. One-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-5, DOC file.
Table 5-6
Intragroup (MCAPT) comparison of Skeleton analysis in different regions of the cortex. One-way repeated-measures ANOVA followed by Tukey’s test. Colored cells indicate p-values < 0.05. Download Table 5-6, DOC file.
In this issue
