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. 2017 Jun 27;7(1):4299.
doi: 10.1038/s41598-017-04082-0.

Long-term Cilostazol Treatment Reduces Gliovascular Damage and Memory Impairment in a Mouse Model of Chronic Cerebral Hypoperfusion

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Free PMC article

Long-term Cilostazol Treatment Reduces Gliovascular Damage and Memory Impairment in a Mouse Model of Chronic Cerebral Hypoperfusion

Akihiro Kitamura et al. Sci Rep. .
Free PMC article

Abstract

Chronic cerebral hypoperfusion is a major cause of age-related vascular cognitive impairment. A well-characterised mouse model has shown that hypoperfusion results in gliovascular and white matter damage and impaired spatial working memory. In this study, we assessed whether cilostazol, a phosphodiesterase III inhibitor, could protect against these changes. Adult, male C57Bl/6J mice were subjected to bilateral common carotid artery stenosis or a sham operation and fed normal or cilostazol diet for three months. Cilostazol treatment reduced the impairment in working memory and white matter function after hypoperfusion. Endothelial adhesion molecules and gliosis, increased after hypoperfusion, were ameliorated with cilostazol treatment. Interestingly, the improvement in working memory was closely correlated with reduced microglia and endothelial adhesion molecules. Further, the number of stroke lesions after hypoperfusion was reduced in the cilostazol-treated group. Altogether cilostazol showed potential to ameliorate the gliovascular damage and working memory impairments after hypoperfusion possibly via endothelial protection supporting its potential use in the treatment of vascular cognitive impairment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cilostazol improved spatial working memory and white matter function: (a) Spatial working memory was assessed using an 8-arm radial arm maze. Statistical analysis of the number of revisiting errors and novel entries indicated an overall significant difference between the 3 groups (F (2, 34) = 5.169; p = 0.029 and F (2, 34) = 5.718; p = 0.022, respectively). Hypoperfused-control mice exhibited significantly more revisiting errors and less novel entries than the sham operated mice (p < 0.05 and p < 0.05), while hypoperfused-cilostazol mice showed a learning process similar to that of sham mice before they plateaued. Data represents the average of 4 trials for block 1 and 2 and the average of 2 trials for blocks 3–6. (b) In the corpus callosum, evoked compound actions potentials (CAPs) were recorded at various distances from the stimulating electrode. Peak latencies of CAPs were significantly different between the three groups (F (2,12) = 14.78; p = 0.0048). The peak latency of CAPs in hypoperfused-control mice was overall significantly increased compared to sham mice (p < 0.01) but this was ameliorated with cilostazol treatment. Data represents mean ± SEM. **p < 0.01, Hypoperfused-control vs Sham.
Figure 2
Figure 2
Cilostazol did not significantly restore white matter integrity. (a) There was no significant difference in Fractional Anisotropy (FA) in the corpus callosum between the three groups. (b) A significant relation between FA and revisiting errors was determined (Spearman r = −0.583, p = 0.0003). (c) MAG grading, as an index of breakdown in axon-glial integrity, was significantly increased after hypoperfusion, but no protective effect of cilostazol could be observed. Data represents mean ± SEM. **p < 0.001, Hypoperfused-control vs Sham; **p < 0.05, Hypoperfused-Cilostazol vs Sham.
Figure 3
Figure 3
Cilostazol reduced the extent of gliosis. (a) Representative images of Iba1 staining in the corpus callosum. (b) There was a trend towards an increase in Iba1 stained micoglia that was reduced with cilostazol after hypoperfusion. There was a robust association between microgliosis and the number of revisiting errors (Pearson r = 0.712, p < 0.0001). (c) Representative images of immunofluorescence for Collagen IV, AQP4 and GFAP in the corpus callosum. (d) There was an increase in the area of GFAP positive astroglia in the hypoperfused-control mice. There was a significant reduction with cilostazol treatment as compared to control hypoperfused. Astrocyte-endfoot displacement tended to be observed in the hypoperfused-control mice and less in the cilostazol treated mice. Data represents mean ± SEM. *p < 0.05, Hypoperfused-control vs Hypoperfused-cilostazol.
Figure 4
Figure 4
Cilostazol significantly suppressed endothelial adhesion molecule expression in the corpus callosum. (a) Representative images of intercellular adhesion molecule-1 (ICAM1) staining in the corpus callosum. (b) There was a significant difference in ICAM staining between the three groups (F (2, 33) = 13.19; p < 0.0001). ICAM1 immunostaining was significantly greater in the hypoperfused-control group compared to the sham (p < 0.001) and this was reduced with cilostazol treatment (p < 0.01). (c) There was a significant association between ICAM1 positive areas and microgliosis (Pearson r = 0.375, p < 0.05), and the number of revisiting errors (Pearson r = 0.412, p < 0.05). Data represents mean ± SEM.
Figure 5
Figure 5
Cilostazol did not affect cerebral blood flow. (a) Representative images of ASL at the level of hippocampus. (b) There was an overall difference in CBF between the three groups in the corpus callosum (F (2, 25) = 11.0; p = 0.0004) and thalamus (F (2, 25) = 33.05; p < 0.0001). CBF was significantly reduced in hypoperfused-control and hypoperfused-cilostazol mice compared to sham mice in the corpus callosum (p < 0.001 and p < 0.001) and the thalamus (p < 0.001 and p < 0.001). Data represents mean ± SEM.
Figure 6
Figure 6
Cilostazol reduced stroke injury. (a) Representative images of T2-MRI detecting the ischemic (arrow head) and hemorrhagic (arrow) lesions. (b) MRI analysis showed that cilostazol remarkably decreased the number of the ischemic stroke lesions after chronic hypoperfuson.
Figure 7
Figure 7
A proposed mechanism by which cilostazol may improve cognitive impairment induced by chronic cerebral hypoperfusion. Cilostazol reduced the number of stroke lesions and significantly ameliorated the spatial working memory impairment and white matter dysfunction induced by chronic cerebral hypoperfusion. Remarkably, cognitive deficits seem to be closely associated with the hypoperfusion-induced neuroinflammatory processes which are significantly decreased with cilostazol treatment. Cilostazol might restore endothelial dysfunction without apparent BBB disruption leading to recovery of neuroinflammation and white matter dysfunction (large arrow).

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