4.6.1. Observation of Natural Changes of Cerebrovascular Amyloid Burden and Smooth Muscle Cell Loss

To confirm the findings of the age-related progression of cerebrovascular amyloid burden and accompanying smooth muscle cell loss in Tg2576 mice, mice fed with standard pelleted chow (i.e., control group) were evaluated at different times (15 months old and 23 months old) using confocal microscopy with double-labeling for Aβ and smooth muscle cell actin. We also evaluated with negative controls without primary antibody for Aβ (follow the same staining protocol without the addition of a primary antibody) to dismiss a possible age-dependent nonspecific stain for the secondary antibody.

#### 4.6.2. Specimens and Raters

All findings were evaluated through the cortex and the hippocampus of the right hemisphere. The ratings of hemorrhagic findings of the brain and any Aβ-positive vessels were evaluated by two raters (K.K. and K.U.), who were blinded to our hypothesis and information of the food content on each mouse. If results were different between the raters, the final decision was made after discussions by the two raters. CAA severity, described below, was classified by a single rater (K.K.) after the determinations of Aβ-positive vessels. Regarding senile plaque, quantitative analysis was performed with a semiautomatic computer-assisted processing system, as mentioned below, by a single rater (K.K.).

#### 4.6.3. Quantitation of Cerebral Hemorrhage(s)

Any acute subdural or cerebral bleeding(s) was defined as a large accumulation of erythrocytes in the intracranial space observed on the H&E stains with sets of systematically sampled sections (every 10th section throughout the cortex and the hippocampus (right hemisphere only)). Cerebral micro-hemorrhages, defined as clusters of hemosiderin staining on Perls's Berlin blue stain with a delayed appearance of hemosiderin-positive microglia [35], located in the brain parenchyma and the around the vessel walls (Figure 3), were quantified on additional sets of every 10th section (right hemisphere only). The ratings of these findings were evaluated by two raters (K.K. and K.U.), who were blinded to our hypothesis and information of the food content on each mouse.

#### 4.6.4. Quantitative Analysis of CAA Burden

All quantification of CAA burden was done as previously published [31]. The frequency and severity of CAA were quantified on systematically sampled serial pan-Aβ immunostained sections throughout the region of interest (every 10th section through the cortex, and the hippocampus). Severity of CAA was classified by a single rater (K.K.), who were blinded to our hypothesis and the information of the food content on each mouse. "CAA frequency" was calculated by counting the total number of any Aβ-positive vessels in the entire set of systematically sampled sections. Regarding the CAA severity, all Aβ-positive vessels were classified into three grades (Figure 5) with a rating scale as described previously [36,37]: severity grade 1 = vessels with a thin rim of amyloid in the vessel wall; severity grade 2 = vascular amyloid with amyloid infiltrating the surrounding neuropil; severity grade 3 = dysphoric amyloid with amyloid deposition within the vessel wall and with a thick and complete amyloid coat around the vessel wall. The mean for all Aβ-positive vessels was taken as CAA severity. To evaluate comprehensive CAA burden, a "CAA score" was calculated by multiplying CAA frequency with CAA severity [31].

#### 4.6.5. Quantitative Analysis of Senile Plaque

Using a computer-assisted processing system (Image J version 1.49 for Mac; National Institutes of Health, Bethesda, MD, USA), the area of pan-Aβ stained lesions in the cortex and the hippocampus, corresponding to senile plaques, was quantified semi-automatically [38] (Scheme A1: please see appendix) by a single rater (K.K.), who was blinded to our hypothesis and the information of the food content on each mouse. Every section of the right hemisphere with Aβ stained electrically was converted to Joint Photographic Experts Group (JPEG) images with the same scale. These JPEG images were analyzed with Image J (version 1.49 for Mac: National Institutes of Health, Bethesda, MD, USA) with appropriate calibrations, as follows. Measurement of area of the section: (1) fill section with red color; (2) dichotomization of color for black and white using a semiautomatic method with an appropriate color threshold; (3) measurement of the black area. Measurement of total area of senile plaque: (1) digital stripping of Aβ-stained lesions located out of the regions of interest; (2) change color of the remaining Aβ-stained area to red using manual calibration with appropriate color threshold; (3) dichotomization of color for black (Aβ-stained lesions in regions of interest (i.e., cortex or hippocampus)) and white (other) using a semiautomatic method with an appropriate color threshold; (4) measurement of the black area. Thus, the total areas of the section, as well as total areas of Aβ-stained lesions, in the right hemisphere were quantitatively measured to count the pixels with a given intensity. To evaluate the degree of the senile plaque in brain parenchyma (including cortex and hippocampus), the percentage of senile plaque area in the brain (% area of senile plaque) was calculated with the following formula:

$$\text{"\(\%\)}\text{ serile plaque = total area of serile plaque / total area of the sections}\tag{1}$$

#### 4.6.6. Statistical Analyse

All statistical analyses were performed with two group comparisons (control group vs. aspirin group or cilostazol group) using the IBM SPSS statistics software program, version 21.0 (IBM, Armonk, NY, USA). As our variables (including cerebral micro-hemorrhages number, CAA score, and percent area of senile plaque) did not follow a normal distribution, a non-parametric test (Mann–Whitney *U* test) was used for the two group comparisons. Log-rank test was used to compare survival rates. *p* values < 0.05 were considered statistically significant.

#### **5. Conclusions**

The present study shows pathological evidence that CAA burden is reduced by cilostazol, even at a low dose. Cilostazol may provide a novel, promising therapeutic target for patients with CAA and/or Alzheimer's disease, potentially in combination with early Aβ immunization therapy.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/7/2295/s1.

**Author Contributions:** Conceptualization, Y.Y. and H.H.; methodology, Y.Y., S.A. and Y.T.; software, Y.Y., K.K.; validation, K.K. and K.U.; formal analysis, K.K. and K.U.; investigation, Y.Y. and K.K.; resources, Y.Y. and H.H.; data curation, Y.Y., Y.N. and K.S.; writing—original draft preparation, Y.Y.; writing—review and editing, M.I. and H.H.; visualization, Y.Y. and K.K.; supervision, H.H.; project administration, Y.Y.; funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Otsuka Pharmaceuticals, which provided the experimental animals purchase cost and the feed.

**Acknowledgments:** We wish to express thanks to Tetsuro Ago (Department of Medical and Clinical Science, Graduate School of Medical Science, Kyusyu University), Ataru Nishihara (Department of Neurosurgery, Graduate School of Medical Science, Kyusyu University), and Naoaki Oyama (Department of Stroke Medicine, Kawasaki Medical School) for their technical advice.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**

