Adult male Sprague Dawley rats (280 to 300 g) and C57/BL6 mice (18 to 25 g) were from Sankyo Labo Service Corporation (Tokyo, Japan). The P2Y₁KO (-/-) (C57/BL6 background) mice were generated previously 11 . All animals were housed in individual cages with a 12-h light/dark cycle and access to food and water ad libitum. Experimental procedures were carried out in accordance with animal experimentation protocols approved by the Animal Care and Use Committee of the University of Tokyo.

Rats and mice were anesthetized intramuscularly with 100 mg/kg ketamine hydrochloride and 25 mg/kg xylazine. Rectal temperature was monitored continuously by a thermometer and heating pads (BWT-100, Bio Research Center, Nagoya, Japan) were used to automatically maintain body temperature at 37.0 to 37.5°C.

For rat MCAO, transient focal cerebral ischemia was induced by a 90-min occlusion of the right middle cerebral artery, as described previously 26 . In brief, a 4-0 nylon monofilament (Nitcho Kogyo Co., Ltd., Tokyo, Japan) with a silicon-coated tip was introduced into the right internal carotid artery from the common carotid artery, and the filament tip was kept in place for 90 min. The filament was then withdrawn from the internal carotid artery to allow reperfusion. After good spontaneous breathing was confirmed, each animal was returned to its cage. For the sham-operated rats, the common carotid artery and the right external carotid artery were occluded.

For mouse MCAO, 8-0 nylon with an expanded (heated) tip was introduced into the right internal carotid through the external carotid stump, as described previously 27 . The infarction time was 45 min. To evaluate the infarcted area, serial coronal sections were obtained from sites +3, +2, +1, +0, -1, -2, and -3 mm from the bregma. The sections were stained with 0.5% cresyl violet and scanned on both sides using a digital camera (Camedia C-5050, Olympus Corporation, Tokyo, Japan). For the continuous administration of MRS2500 (Tocris Bioscience, Minneapolis, MN, USA) into mice, a micro-osmotic pump (model 1007D, Alzet, Palo Alto, CA, USA) filled with 100 μL MRS2500 (1 mg/mL), delivering 0.5 μL/h, was implanted intracerebroventricularly immediately after reperfusion using a Brain Infusion Kit 3 (Alzet, Palo Alto, CA, USA).

To evaluate cognitive function, mice and rats were subjected to fear conditioning 24 h before contextual and cued tests. For conditioning, rats were placed individually in a conditioning chamber (Med Associates, Inc., St Albans, Vermont, USA) for 5 min; after 3 min they were given two tone/foot-shock pairings. A 10-s tone (80 dB, 5 KHz) preceded a 2-s foot-shock (0.75 mA) that co-terminated with the tone (60-s interstimulus interval). Mice were placed in a box for 6 minutes and given three tone/foot-shock pairings (1 mA).

For both mice and rats, the chambers were inside a sound-attenuating box equipped with a fan as white noise, to minimize the effect of outside noise. The box was cleaned with 70% isopropanol before each use. For contextual testing, freezing behavior was scored for 8 min without any stimulation. For cued testing, the animals were placed in a different context (red lighting, flat floor, and curved wall) for 5 min following the contextual test. After 3 min, a 10-s tone was delivered twice using the same protocol as in the conditioning, without the shock. The animals’ freezing behavior was scored for 3 min before the tone (pre-tone) and for 2 min after the tone was delivered (post-tone).

A 21-point behavioral scale 28 was used to evaluate the sensory-motor function of rats after MCAO. Sham and MCAO groups were prepared specifically for the daily behavioral experiments (n = 6 each). However, when mice were used for these tests, we could not evaluate their standing ability on a 45-degree slope as described by Hunter 28 , because they weighed too little for the test to be executed correctly.

Following the behavioral tests, the rats were sacrificed at three time points: 1 week, 3 weeks, and 2 months after surgery (only at 1 week for sham-operated animals). They were perfused with phosphate-buffered saline (PBS) intracardially and decapitated. Each brain was post-fixed in 4% formaldehyde (from paraformaldehyde) in PBS (pH 7.4) for 30 min at 4°C. The fixed brain was rinsed with PBS and was then placed into the scanner within a plastic tube.

The magnetic resonance (MR) experiments on rats were performed in a 4.7 T scanner (Varian Associates, Palo Alto, CA) equipped with gradients of up to 60 mT/m. A 66-mm volume coil was used for both transmission and reception. A 2D diffusion-weighted (DW) spin-echo sequence was used with the following acquisition parameters: FOV = 30 × 30 mm², image matrix = 128 × 128, slice thickness/gap = 1/0 mm, TR/TE = 3000/50 ms, NEX = 40. DW images were acquired in 12 directions with 2 b-values of 0 and 1496 s/mm². The total acquisition time was 55 h.

Mice were perfused intracardially with PBS, then with 4% formaldehyde in PBS (pH 7.4), and decapitated. Each brain was post-fixed in 4% formaldehyde for at least 3 days at 4°C. The fixed brain was rinsed with PBS and immersed in Fluorinert (Sigma Aldrich, St. Louis, MO, USA) in a glass tube, because the MRI system is vertical. The mouse MR experiments were performed in a 14.1 T scanner (Bruker BioSpin, Billerica , MA, USA) equipped with gradients of up to 3000 mT/m. A 10-mm volume coil was used for both transmission and reception. A 2D diffusion-weighted (DW) spin-echo sequence was used with the following acquisition parameters: FOV = 6 × 6 mm², image matrix = 128 × 128, slice thickness/gap = 0.5/0 mm, TR/TE = 2500/27 ms, NEX = 16. DW images were acquired in 12 directions with 2 b-values of 0 and 1500 s/mm². The total acquisition time was 19 h.

For the DTI of both rats and mice, motion-probing gradients (MPGs) were applied as follows:

g 1 = 5 5 201 , g 2 = 5 5 012 , g 3 = 5 5 120 , g 4 = 5 5 210 , g 5 = 5 5 021 , g 6 = 5 5 102 , g 7 = 5 5 20 ‒ 1 , g 8 = 5 5 012 , g 9 = 5 5 ‒ 120 , g 10 = 5 5 2 ‒ 10 , g 11 = 5 5 02 ‒ 1 and g 12 = 5 5 ‒ 102

These sample preparation and DTI data acquisition methods were based on previous research with minor modifications 19 29 .

The six independent elements of the 3 × 3 diffusion tensor were calculated from each series of DW images. The tensor was diagonalized to obtain three eigenvalues (λ_1-3), which corresponded to the three eigenvectors (ν_1-3). The DTI index Trace(D), which is a scalar measurement of the total diffusion within a voxel, was derived from the DTI-studio software 30 .

Trace D = λ 1 + λ 2 + λ 3

The mean diffusivity (MD) is a measure of the average motion of water molecules independent of directionality, which was obtained from the Trace(D). For the color-coded MD maps, MATLAB (Math Works, Natick, MA, USA) was used. The MD value range was defined as 0 to 5.0 × 10^-4 mm²/sec.

MD = λ 1 + λ 2 + λ 3 3 = Trace D 3

Regions of interest (ROIs) were semi-automatically defined in the images generated from DTI Studio, by ROI Editor 30 .

To make the T-contrast map, SPM5 software (Welcome Trust Center for Neuroimaging, England, UK) running on MATLAB, was used for slice realignment and spatial normalization of the DTI data 31 . We analyzed the areas that showed a significant decrease in MD (P <0.001, uncorrected) at 1 week after MCAO (n = 6), compared with intact animals (n = 7).

For fluorescence immunohistochemistry, frozen specimens were coronally sectioned with a cryostat (Microm) at a thickness of 40 μm. After being blocked in 3% donkey serum, the sections were incubated with anti-glial fibrillary acidic protein (GFAP; mouse, 1:800; Sigma Aldrich, St. Louis, MO) and anti-ionized calcium binding adaptor molecule 1 antibodies (Iba1; rabbit, 1:1000; Wako) overnight at 4°C. The secondary antibodies were Alexa 488-conjugated donkey anti-rabbit IgG (1:1,000; Molecular Probes) and Cy5-conjugated donkey anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The stained sections were incubated with DAPI (Sigma Aldrich), and observed using a confocal laser microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Images obtained via confocal microscopy were analyzed with ImageJ software, as reported in our previous study 32 . A custom plugin was used to automatically establish the overall fluorescence from the image (> 0.28 mm²) at the dentate gyrus (DG)/hilus, the CA2 and CA3 (CA2/3) regions, or the CA1 region, and to calculate the percentage of the image covered by staining.

The data are shown as the mean ± SEM. Statistical significance was determined using Student’s t-test and Tukey’s multiple comparison test. P <0.05 indicated statistically significant differences.

We tested the cognitive function of rats 1 week, 3 weeks, and 2 months after MCAO, and observed a clear decrease in the freezing time in the contextual conditioned test at all the time points tested, compared to the control rats 1 week after the sham operation (n = 6 in each group; P <0.05, Figure 1A). In contrast, in a cue test, we did not observe a significant difference in the freezing behavior among the groups (data not shown).

We also evaluated the time course of sensory-motor function after MCAO. A sensory-motor neurological test was conducted in a different cohort of MCAO and sham-operated rats (n = 6 in each group) every day from 1 to 28 days after the operation. As shown in Figure 1B, we detected significant recovery in the sensory-motor function of the MCAO rats, suggesting the regeneration and/or functional repair of the damaged neural networks. Taken together, these findings indicated that over the long term after MCAO, the occurrence of cognitive deficits was still a concern, despite sensory-motor recovery.

For the sensitive detection of microstructural alterations in the hippocampus of MCAO rats, we performed ex-vivo DTI analyses. The brain was fixed with 4% formaldehyde for 30 min at 4°C, by a slight modification of the method described by Shepherd 17 . The post-fixed brain was placed in a plastic tube without any solution and scanned in a 4.7-T animal magnetic resonance imaging (MRI) with 12-axis multi-slice DTI for 55 hours. We analyzed the change in the affected hippocampus by determining the mean diffusivity (MD) value, a type of DTI index. Examples of coronal color images of the MD values in Figure 2 show the brain from a normal non-operated rat as an intact control, the brain from a sham-operated rat 1 week after surgery as an internal control, and the brains of MCAO-treated rats 1 week, 3 weeks, and 2 months after surgery. A detailed visual inspection revealed a clear decrease in the MD value at the CA2/3 pyramidal cell layer of the ipsilateral hippocampus (indicated by an arrowhead in Figure 2C).

Next, we quantitatively analyzed the change in the MD value at the affected hippocampus. For this analysis, we used DTI Studio for the MD calculation, with semi-automatic morphometric identification of the hippocampus by ROI Editor. We detected a significant reduction in the MD value of the ipsilateral hippocampus (P <0.05) after MCAO, compared to the intact or sham group (Figure 3C). The contralateral hippocampus did not show a significant MD decrease after MCAO (data not shown). For a more precise verification of the decreased MD value, we performed a voxel-based morphometric (VBM) analysis, as a fully automatic digital comparison of imaging data between the MCAO and sham-operated rats. The T-contrast map image revealed a significant reduction in the MD value after MCAO (Figure 3D).

To evaluate the neuroinflammation-related cellular changes in the ipsilateral hippocampus after MCAO, we stained brain sections with anti-GFAP (a marker for activated astrocytes) and anti-Iba1 (a marker for activated microglia). As shown in Figure 4, separate subregions of the hippocampus were scanned (DG/hilus, CA2/3, and CA1). Increased activation of both astrocytes and microglia was observed in the CA2/3 region (Figure 4B, D), suggesting that neuroinflammatory responses were evoked in the hippocampus by MCAO. These alterations were also observed in the hippocampal slices 3 weeks and 2 months after MCAO (data not shown).

To examine the molecular basis of the cognitive deficits subsequent to MCAO, we used P2Y₁KO mice and wild-type (WT) C57BL/6 mice. We prepared four groups of mice: WT-sham, WT-MCAO, P2Y₁KO-sham, and P2Y₁KO-MCAO. We first measured the cognitive and sensory-motor function 1 week after MCAO. In the contextual conditioning test, the WT mice showed cognitive deficits, as seen in rats. However, the P2Y₁KO mice exhibited no decline in cognitive function after MCAO (Figure 5A). The P2Y₁KO-sham mice exhibited cognitive function equivalent to that in WT-sham mice. The sensory-motor function was equally impaired in the WT-MCAO and P2Y₁KO-MCAO mice (Figure 5B). The size of the infarction at the neocortex and the striatum as evaluated by Nissl staining was also equivalent between the two groups (WT: 13.6 ± 1.37%; KO: 12.1 ± 1.70% of the total brain).

We stained mouse brain sections in the same manner as in the study of rats. Images from the ipsilateral hippocampus of the WT-MCAO and P2Y₁KO-MCAO groups were evaluated (Figure 6). In the P2Y₁KO mice, the activation of astrocytes was decreased in the CA2/3 region (Figure 6B), and significantly fewer activated microglia were seen in all the regions (Figure 6D) compared with wild-type mice, suggesting that the neuroinflammatory responses were suppressed in the hippocampus of the P2Y₁KO mice. We also checked the background glial-activation level in the sham-operated P2Y₁KO mice, and found no difference between the KO-sham and WT-sham groups (data not shown).

To examine the involvement of P2Y₁-mediated signaling in the course of the cognitive decline induced by a focal cerebral ischemia, MCAO, we examined whether cognitive decline occurred 3 to 4 days after the operation, during a more acute phase of the ischemic injury. We chose this time point because P2Y₁-dependent neural stem cell proliferation was not pronounced at 3 days, compared to 1 week after MCAO (data not shown); therefore, the putative P2Y₁-dependent cognitive decline might not occur until the 1-week time point.

For this experiment, we utilized a P2Y₁-specific antagonist MRS2500 and prepared five groups of mice: WT-sham, WT-MCAO, WT-MCAO-MRS2500, P2Y₁KO-sham, and P2Y₁KO-MCAO. Contrary to our initial speculation, in the contextual conditioning tested 4 days after injury, the WT-MCAO mice showed significant cognitive deficits compared to the WT-sham mice. Interestingly, WT-MCAO mice treated with the P2Y₁ antagonist MRS2500 by osmotic pump soon after reperfusion, did not show any cognitive decline. In addition, the P2Y₁KO-MCAO mice exhibited cognitive function equivalent to that of the WT-sham and P2Y₁KO-sham mice (Figure 7). Taken together, these findings suggest that P2Y₁-mediated signaling from the time of reperfusion to 4 days after MCAO contributes to the MCAO-induced cognitive decline in mice.

We next performed an ex-vivo DTI analysis using the brains of mice that had been used for the behavioral test shown in Figure 7. Within a day after the behavior test, the brains were fixed with 4% formaldehyde for at least 3 days at 4°C. The post-fixed brains were placed into 10-mm NMR tubes and scanned in a 14.1-T animal MRI with 12-axis multi-slice DTI for 19 hours. We analyzed the change in the affected hippocampus by determining the MD value after DTI. We detected a significant reduction in the MD value of the ipsilateral hippocampus (P <0.05) of the WT-MCAO mice compared to the WT-sham mice (Figure 8). In contrast, the P2Y₁KO-MCAO brains showed a hippocampal MD value equivalent to that of the WT-sham and P2Y₁-sham brains. A detailed visual inspection revealed a clear decrease in the MD value at the CA2/3 pyramidal cell layer of the ipsilateral hippocampus of the WT-MCAO brains, compared to that of the P2Y₁KO-MCAO brains (indicated by arrowheads in Figure 8D, E).