Clopidogrel Administration Impairs Post-Stroke Learning and Memory Recovery in Mice

Figures

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  Figure 1. PAL task performance post-stroke. (A) A schematic depicting the stimuli used during the PAL task (flower, airplane, spider), which were only correct when displayed in the left (L), middle (M), or right (R) locations, respectively. (B) A schematic depicting six different types of trials (1–6) that occurred during the PAL task. Two different stimuli were presented in each trial: one was presented in its correct location (S+) while the other was presented in an incorrect location (S−). (C) The graph shows the percentages of correct responses of mice in each of the three groups (sham, stroke + control, stroke + clopidogrel) in the first and second weeks of treatment. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 2. The effect of short-term clopidogrel administration on motor impairment. (A) Illustrates the placement of the mouse in the cylinder in which the spontaneous forelimb placement task was undertaken. (B) Cylinder task asymmetry scores for the three groups (sham, stroke + control, stroke + clopidogrel) at baseline (one day before stroke (/sham) surgery) and at 2, 7, and 14 days post-stroke. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated. ^ denotes changes between the sham-operated and stroke + control animals, # denotes changes between the sham-operated and stroke + clopidogrel animals, and * denotes changes between the stroke + control and stroke + clopidogrel animals.

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  Figure 3. The effect of short-term clopidogrel administration on physiological parameters. (A) The survival rate and (B) body weight were recorded daily for 14 days post-stroke for the sham-operated mice, stroke + control-treated mice, and stroke + clopidogrel-treated mice. The body weight change was calculated as a percentage (%) change from day 0. The mouse survival rates were analyzed using a simple survival analysis (Kaplan–Meier). Mouse body weight data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated. ^ denotes changes between the sham-operated and stroke + control animals, # denotes changes between the sham-operated and stroke + clopidogrel animals, and * denotes changes between the stroke + control and stroke + clopidogrel animals.

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  Figure 4. The effects of short-term clopidogrel treatment on vasculature within the ipsilateral and contralateral cortexes. Collagen IV labeling within (A) the ipsilateral cortex region and (B) the contralateral cortex region (scale bar = 80 µm). The top images show the regions examined and representative collagen IV labeling observed for the three groups: sham, stroke + control, and stroke + clopidogrel. The bar graphs underneath show changes in the total area covered by vessels and the number of vessels within the regions examined. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 5. The effects of short-term clopidogrel treatment on vascular leakage in the ipsilateral and contralateral cortexes. (A) The location of the ipsilateral and contralateral cortex regions examined and representative labeling of IgG for the three groups: sham, stroke + control, and stroke + clopidogrel (scale bar = 60 µm). The bar graphs show the quantification of IgG thresholding within (B) the ipsilateral cortex and (C) the contralateral cortex. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 6. The effects of short-term clopidogrel treatment on the microglia number and morphology within the ipsilateral and contralateral cortexes. Iba1 labeling within (A) the ipsilateral cortex region and (B) the contralateral cortex region (scale bar = 60 µm). The top images show the regions examined and representative labeling observed for Iba1 for the three groups: sham, stroke + control, and stroke + clopidogrel. Microglia were reconstructed based on MicroTrac software detection of Iba1 labeling then, the microglia number and morphology were analyzed. The bar graphs underneath show the changes in the microglia number and soma area within the regions examined. All cells within one image were individually reconstructed and morphological parameters were averaged for each image, one per animal. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 7. The effects of short-term clopidogrel treatment on neurons within the peri-infarct region. The location of the peri-infarct region examined and representative labeling of (A) NeuN-positive cells (scale bar = 65 µm) and (B) fibrinogen-positive cells (scale bar = 55 µm) for the three groups: sham, stroke + control, and stroke + clopidogrel. The bar graphs show the quantification of (C) the number of NeuN-positive cells and (D) the number of fibrinogen-positive cells. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 8. The effects of short-term clopidogrel treatment on the T cell number within the ipsilateral and contralateral cortexes. CD3 labeling within (A) the ipsilateral cortex region and (B) the contralateral cortex region (scale bar = 60 µm). Images show the regions examined and representative CD3 labeling observed for the three groups: sham, stroke + control, and stroke + clopidogrel. The bar graphs show the quantification of the CD3 labeling within (C) the ipsilateral cortex and (D) the contralateral cortex. Data were checked for normality using the Shapiro–Wilk normality test and then analyzed using two-way ANOVA with multiple comparisons (Benjamini, Krieger, and Yekutieli test). Data are given as the mean ± SEM. Significant p-values are indicated.

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  Figure 9. Experimental design timeline. (A) Photothrombotic occlusion set-up. (B) The first cohort of mice underwent motor impairment assessment using the cylinder task and memory and learning assessment using the PAL task. (C) The second cohort of mice was used for fixed tissue analysis (immunohistochemistry).

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  Figure 10. Touchscreen training. (A) Must-touch training phase. The mice had to complete 36 trials within 60 min during the must-touch training phase before proceeding to the next phase. (B) Punish-incorrect training phase. The mice had to attain a correct rate of 75% in the punish-incorrect training phase before commencing the PAL task.