Introduction
Defects in hippocampal function caused by the progression of cerebrovascular disorders or Alzheimer’s disease can lead to memory loss. Identifying the molecular mechanisms underlying these cognitive deficits is a long-standing goal in neuroscience. Recent studies suggest that neuroinflammation may directly cause hippocampal network dysfunction in the aging brain [
1,
2]. Although neuroinflammation inducers, including beta-amyloid protein, hypoxia, and oxidative stresses, are generated in individual microenvironments [
2], their effects converge in the downstream molecular and cellular cascades that transduce neuroinflammatory responses ( for example, microgliosis or astrogliosis) [
3]. A strong association between such glial activation and changes in the hippocampal microenvironment, has been shown in mouse models of cerebral ischemia and Alzheimer’s disease [
4,
5]. The inflammatory responses generated by the activated astrocytes disturb synaptic plasticity and may cause a decline in cognitive function [
6,
7].
The glial neuroinflammatory response is known to involve the ADP/ATP receptor P2Y
1, which is widely distributed throughout the central nervous system (CNS). The P2Y
1 receptors are upregulated in glial cells in various brain regions after ischemia [
8]. Thus, P2Y
1 on astrocytes may transduce neuroinflammatory responses when activated by ATP released from damaged neurons or glial cells in various CNS disorders [
9]. In addition, astrocytic P2Y
1 activation induces the release of proinflammatory cytokines [
10]. Thus, it is important to examine P2Y
1’s role under pathological conditions. Here we used P2Y
1-knockout (P2Y
1KO) mice, which were recently reported to resist vascular inflammation [
11,
12]. We subjected the P2Y
1KO mice to middle cerebral artery occlusion (MCAO) to assess P2Y
1’s role in initiating the neuroinflammatory response in the affected hippocampus and the consequences of the neuroinflammation, including cognitive deficits. The rodent MCAO model causes both sensory-motor and cognitive deficits [
13,
14]. For example, rats subjected to MCAO perform worse than sham-operated rats in the Morris maze task [
15]. Although the neural atrophy caused by MCAO is primarily observed in the striatum and cerebral cortex, previous studies have reported conflicting results regarding the occurrence of hippocampal damage following MCAO [
13,
15‐
18].
To monitor putative hippocampal microstructural changes relating to the neuroinflammation caused by MCAO, we applied a sensitive neuroimaging method, diffusion tensor MRI (DTI) [
19,
20]. DTI can detect microscopic structural changes that alter water diffusivity, such as the enlargement of glial cells upon their activation, in a region of neuroinflammation [
21,
22]. This technique previously revealed the magnitude of water diffusivity alteration in the rat hippocampus after two-vessel occlusion [
23] and in the mouse hippocampus in an Alzheimer’s disease model [
24,
25].
In this study, we focused on the hippocampal environmental changes following the neuroinflammatory response induced by MCAO. We examined glial activation by immunohistochemistry and structural changes by DTI. The aim of this study was to discover how an alteration in the hippocampal microenvironment affects cognitive function after MCAO. We also focused on the role of the astrocytic P2Y1 receptor in regulating the neuroinflammatory response that affects cognitive function.
Material and methods
Animals
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 P2Y
1KO (-/-) (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.
Surgery
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).
Behavioral tests
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.
Diffusion tensor imaging data acquisition
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:
These sample preparation and DTI data acquisition methods were based on previous research with minor modifications [
19,
29].
Image and data analysis
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].
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 mm
2/sec.
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).
Immunohistochemical analysis
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 mm
2) 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.
Statistical analysis
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.
Discussion
The cognitive impairment that occurs after cerebral ischemia, especially in memory function, has recently attracted much attention [
14‐
16,
34,
35]. In animal studies, MCAO disturbs cognitive function as detected by a water maze test [
14,
15] or a contextual conditioning test (this study). However, to our knowledge, there are few reports monitoring the chronic pathological or histological alterations in the hippocampus after MCAO. In this study, we observed impaired cognitive function, hippocampal neuroinflammation, and microstructural changes in MCAO model rats and mice. To examine the underlying mechanism affecting cognitive function, we focused on the ADP/ATP receptor P2Y
1 expressed on activated glial cells. Our findings clearly indicated that P2Y
1-dependent neuroinflammation contributes to the pathophysiological mechanism of the functional alteration in the hippocampus.
In this study, we successfully depicted stroke-related hippocampal microstructural alterations by an MRI method, diffusion tensor MRI (DTI). DTI has been used to detect tissue abnormalities in a variety of diseases [
36‐
39].
Ex-vivo DTI has several advantages over
in-vivo DTI, including better signal-to-noise ratio, improved spatial resolution, and fewer motion artifacts [
19].
Ex-vivo DTI is suitable for animal studies, in which tissue can be removed for detailed evaluation, and may be superior to conventional T2-weighted imaging or immunohistochemical methods. Our results showed that MCAO caused a significant reduction in the hippocampal MD, which may indicate microstructural tissue changes owing to cell swelling or hypertrophy [
36]. We assume that the stroke-related enlargement of activated astrocytes or microglial cells contributes to this putative structural change after MCAO. However, the hippocampal MD value did not recover, which would be related to the long-term deficit (2 months after MCAO) in cognitive function. In both ROI-based analyses using DTI Studio [
30] and group-based analyses using SPM [
31], a significant MD change between the MCAO and sham-operated animals was observed. However, we did not find a significant change in fractional anisotropy value, in contrast to a previous study [
39].
It is reasonable to assume that the reduction in MD value we observed is related to the augmented neuroinflammatory response. We detected a significant elevation of the neuroinflammatory response in the CA2/3 region of the hippocampus by anti-GFAP and anti-Iba1 immunohistochemistry, and this region also showed the most significant reduction in MD value in both the MD color maps and the T-contrast map. Concurrently, we carefully examined the integrity of the CA1 pyramidal cell layer, which is the most vulnerable area in the hippocampus [
40], but did not detect hippocampal neuronal death in our model (data not shown).
Our study opens the possibility that P2Y
1-dependent inflammatory responses are associated with cognitive deficit. Recent studies have shown that ischemia increases the extracellular ATP released by damaged cells, which stimulates astrocytic P2Y
1, resulting in high GFAP expression [
10,
41]. Given that GFAP upregulation occurs 12 h after reperfusion [
42], we also evaluated the cognitive function at a more acute phase (3 to 4 days), and administered a P2Y
1 antagonist, MRS2500, soon after the reperfusion. Our findings clearly showed that P2Y
1signaling has a critical role from the time right after reperfusion through the next 4 days. During these days, it can be supposed that microglia initially reacts to ischemic damage and releases small amount of ATP which augment the P2Y1-mediated signaling on astrocytes, as reported by Pascual
et al. [
43]. Although activated astrocytes sometimes provide beneficial effects by producing cytokines that support cell regrowth [
29,
44], these cytokines, such as TNF-α, interleukin-6, or interleukin1-β, may also disturb synaptic plasticity, such as in LTP and cognitive function [
6,
7,
10,
45‐
47].
In line with this scenario, we clearly demonstrated that the blockade of P2Y
1-mediated signaling, by either P2Y
1KO mice or a P2Y
1-specific antagonist, ameliorated the cognitive deficits induced by a focal cerebral stroke, MCAO. Very recently, Choo
et al. reported that the antagonism of P2Y
1 reduces hippocampal neuronal death and cognitive deficit after traumatic brain injury [
48]. Data from our study suggest that the signaling through P2Y
1 receptors on glial cells contributes to hippocampal neuroinflammation and cognitive deficit, and these findings may lead to new therapeutic strategies for brain infarction, targeting the P2Y
1 receptor.
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
TH, MS, and YC designed the experiments. YC, MK, FN, YA, YT, HO, FK, SK, and CG collected and analyzed data. YC and TH drafted the manuscript with the assistance of the other authors. All the authors read and approved the manuscript.