Role and mechanism of P2Y₁₂ receptor in microglia in central post-stroke pain in mice

Objective To evaluate the role of P2Y₁₂ receptor in microglia in central post-stroke pain (CPSP) in mice and its mechanism.

Methods Thirty-two specific pathogen-free(SPF)-grade male C57BL/6J mice were selected, aged 7 to 8 weeks, weighing 25 to 30 g, were divided into four groups using a random number table method: C group (sham operation group, n=8), CPSP group (n=8), CPSP-M group (CPSP plus P2Y₁₂ receptor inhibitor MRS2395, n=8) and C-M group (sham operation plus P2Y₁₂ receptor inhibitor MRS2395, n=8). CPSP model was induced by injecting 10 nL type Ⅳ collagenase(0.001 U/nL) into the right ventral posterior medial nucleus andventral posterior lateral nucleus. MRS2395(1.5 mg/kg) was injected intraperitoneally at 30 min before model preparation in CPSP-M group and C-M group and then injected once a day for 5 consecutive days, mice in C group and CPSP group were given saline at the same volume. Before the modeling (T₀) and 3, 7 and 14 days (T₁, T₂, T₃) after the establishment of the model, the thermal withdrawal latency (TWL), cold withdrawal latency (CWL) and paw withdrawal frequency (PWF) were measured. The mice were then sacrificed and the brain tissues were obtained for observing the location of P2Y₁₂receptor by double immunofluorescence staining and the pathological changes by HE staining, and for determination of the expression levels of P2Y₁₂ receptor, toll-like receptor 4 (TLR4), phospho-nuclear factor-κB p65 (p-NF-κB p65) and NF-κB p65 by western blot.

Results P2Y₁₂ receptor was co-localized only with microglia, but not with neurons or astrocytes in brain tissue of CPSP group. Compared with C group, the TWL and CWL were significantly reduced and the PWF was significantly increased at T₁ to T₃, cerebral injury was aggravated significantly and the expressions of P2Y₁₂ receptor, TLR4 and p-NF-κB p65 in brain tissue were up-regulated in CPSP group and CPSP-M group (P < 0.05). There was no statistically significant difference between C group and C-M group in the above indicators (P>0.05). Compared with CPSP group, the TWL and CWL were significantly prolonged and the PWF was significantly reduced at T₁ to T₃, cerebral injury was relieved significantly and the expressions of P2Y₁₂ receptor, TLR4 and p-NF-κB p65 in brain tissue were down-regulated in CPSP group and CPSP-M group (P < 0.05).

Conclusion P2Y₁₂ receptors in microglia may be involved in the development of CPSP by activating TLR4/NF-κB signaling pathway in mice.