Involvement of glial P2Y₁ receptors in cognitive deficit after focal cerebral stroke in a rodent model

Abstract

Background: Neuroinflammation is associated with many conditions that lead to dementia, such as cerebrovascular disorders or Alzheimer's disease. However, the specific role of neuroinflammation in the progression of cognitive deficits remains unclear. To understand the molecular mechanisms underlying these events we used a rodent model of focal cerebral stroke, which causes deficits in hippocampus-dependent cognitive function.

Methods: Cerebral stroke was induced by middle cerebral artery occlusion (MCAO). Hippocampus-dependent cognitive function was evaluated by a contextual fear conditioning test. The glial neuroinflammatory responses were investigated by immunohistochemical evaluation and diffusion tensor MRI (DTI). We used knockout mice for P2Y₁ (P2Y₁KO), a glial ADP/ATP receptor that induces the release of proinflammatory cytokines, to examine the links among P2Y₁-mediated signaling, the neuroinflammatory response, and cognitive function.

Results: Declines in cognitive function and glial neuroinflammatory response were observed after MCAO in both rats and mice. Changes in the hippocampal tissue were detected by DTI as the mean diffusivity (MD) value, which corresponded with the cognitive decline at 4 days, 1 week, 3 weeks, and 2 months after MCAO. Interestingly, the P2Y₁KO mice with MCAO showed a decline in sensory-motor function, but not in cognition. Furthermore, the P2Y₁KO mice showed neither a hippocampal glial neuroinflammatory response (as assessed by immunohistochemistry) nor a change in hippocampal MD value after MCAO. In addition, wild-type mice treated with a P2Y₁-specific antagonist immediately after reperfusion did not show cognitive decline.

Conclusion: Our findings indicate that glial P2Y₁ receptors are involved in the hippocampal inflammatory response. The findings from this study may contribute to the development of a therapeutic strategy for brain infarction, targeting the P2Y₁ receptor.

Figure 1

Long-lasting cognitive decline after middle cerebral artery occlusion (MCAO) in rats. Hippocampus-dependent cognitive function was assessed by a contextual fear conditioning test. The freezing time was measured for 8 minutes in MCAO and sham-operated rats (A). Significant differences were seen at all the time points between the MCAO and sham-operated group. Daily neurological testing was conducted in a separate cohort for 4 weeks (B). Most of the sensory motor function of the MCAO rats recovered within the experimental period. n = 6 in each group. Values are means ± SEM. ^*P <0.05; ^**P <0.01; in the t-test.

Figure 2

Alteration in hippocampal mean diffusivity (MD) in middle cerebral artery occlusion (MCAO) rats visualized by MD map. Diffusion-weighted anatomic images of the brains from the intact, sham, and 1-week, 3-weeks, and 2-months after MCAO groups (A). MD color maps were created by MATLAB in the same slice for each group (B). Enlarged images of the boxed hippocampal areas of the sham and 1 week after MCAO brains shown in B (C). The infarcted area shows an unusually high MD value in the ipsilateral side (left) of the brain. Meanwhile, the white arrowhead indicates MD decreasing on the ipsilateral side of the hippocampus. MD values ranged from 0 to 5.0 × 10^-4 (mm²/sec), as shown in the color bar below. All slices were obtained at bregma -3.30 to -3.80 mm.

Figure 3

Region of interest (ROI)-based and voxel-based morphometric (VBM) analyses of the hippocampal mean diffusivity (MD) from middle cerebral artery occlusion (MCAO) rats. The histological images were obtained from the rat brain atlas (A, [33]). Regions of interest (ROI) for the hippocampal area were defined semi-automatically on diffusion-weighted images by ROI Editor (B: bregma -3.30 to -3.80 mm). The hippocampal MD of the ipsilateral side was calculated for each group (C). All the post-MCAO groups showed a significant decrease. n = 6 for each group. Values are means ± SEM. *P <0.05; **P <0.01 in the t-test versus intact group. ^#P <0.05; ^###P <0.001 in the t-test versus sham-operated group. T-contrast map showing a significant decrease in MD value was observed in the 1 week after MCAO group compared to the intact group (D: uncorrected, P <0.001). The ipsilateral hippocampus and striatum areas showed significant differences. For the groups tested at 3 weeks and 2 months after MCAO, the realignment process could not be performed by the software, because the chronic MCAO brains were too greatly altered. Color bars, t value (Height threshold t = 4.025).

Figure 4

Hippocampal neuroinflammatory gliosis after middle cerebral artery occlusion (MCAO) in rats. Representative comparisons between 1 week post-MCAO and sham-operated rats (A,C). Slices from each region of the hippocampus (dentate gyrus (DG)/hilus, CA2/3, and CA1) were stained with anti-GFAP and anti-Iba1 antibodies and scanned. The proportion (area ratio) of GFAP (B) and Iba1 (D) staining was calculated for each region. n = 4 in each group. Red, GFAP; Green, Iba1; Blue, DAPI. Bar represents 100 μm. Values are means ± SEM. *P <0.05; **P <0.01 in the t-test.

Figure 5

Cognitive function was retained in P2Y₁KO mice after middle cerebral artery occlusion (MCAO). Contextual fear conditioning (A) and neurological (B) tests similar to the experiments with rats were performed. Both of the tests were conducted 1 week after MCAO. P2Y₁KO mice retained their hippocampus-dependent cognitive function, while they showed sensory motor deficits similar to WT mice. WT-sham, n = 18; WT-MCAO, n = 19; P2Y₁KO-sham, n = 14; P2Y₁KO-MCAO, n = 14. Values are means ± SEM. **P <0.01, ***P <0.001 in Tukey’s multiple comparison test.

Figure 6

Lack of hippocampal neuroinflammation in P2Y₁KO mice. Representative comparisons between WT-MCAO and P2Y₁KO-MCAO mice 1 week after surgery (A,C). Slices showing each region of the hippocampus (DG/hilus, CA2/3, and CA1) were stained with anti-GFAP and anti-Iba1 antibodies and scanned. The proportion (area ratio) of GFAP (B) and Iba1 (D) staining was calculated for each region. In KO mice, the GFAP expression was suppressed significantly in CA2/3, whereas Iba1 was significantly decreased in all hippocampal areas. Red, GFAP; green, Iba1; blue, DAPI. n = 4 in each group. Values are means ± SEM. *P <0.05; **P <0.01 in the t-test. Magnification = 40×; scale bar, 100 μm.

Figure 7

Cognitive function in the acute phase and effect of blocking P2Y₁. A contextual fear conditioning test was conducted 4 days after middle cerebral artery occlusion (MCAO). The results were similar to those of the 1 week after MCAO group. The P2Y₁ receptor antagonist MRS2500 led to remarkably maintained cognitive function. MRS2500 was administered by pump, starting immediately after the 45-min MCAO. WT-sham, n = 7; WT-MCAO, n = 10; WT-MCAO-MRS2500, n = 5; P2Y₁KO-sham, n = 9; P2Y₁KO-MCAO, n = 8. Values are means ± SEM. **P <0.01, in Tukey’s multiple comparison test.

Figure 8

No decrease in hippocampal mean diffusivity (MD) value after middle cerebral artery occlusion (MCAO) in P2Y₁KO-MCAO mice. Ex-vivo T2-weighted (A) and diffusion-weighted (B) images of the ipsilateral hemisphere. Regions of interest ( ROIs) for the hippocampal area were defined semi-automatically using ROI Editor (B). The MD value of the ipsilateral side of the brain from four groups was calculated (C). WT-sham, n = 4; WT-MCAO, n = 4; P2Y₁KO-sham, n = 4; P2Y₁KO-MCAO, n = 4. Values are means ± SEM. *P <0.05, **P <0.01, in Tukey’s multiple comparison test. Representative hippocampal MD maps from WT-MCAO (D) and P2Y₁KO-MCAO (E) mice. MD maps were depicted by MATLAB. White arrowheads indicate the different MD values between the two groups at the ipsilateral side of the hippocampus. MD values ranged from 0 to 5.0 × 10^-4 (mm²/sec), shown in the bar at right. All slices were obtained at bregma -1.80 to -2.30 mm.