Reduction of pain thresholds and elevation of CGRP after the induction of CM in rats
We established a rat model of CM by continuous infusion of IS into the dura mater for seven days and evaluated the success of the model by the mechanical pain thresholds, thermal pain thresholds, and calcitonin gene-related peptide (CGRP) expression level. The PBS group was used as the control group. There was a significant decrease in the mechanical pain thresholds and thermal pain thresholds of hind paw of the CM group rats from the third day after the infusion of IS(Fig. 1A,1B). In addition, CGRP is considered to be closely related to the pathological mechanism of CM, and its expression level is used as a key index to evaluate the success of the model, so we also detected the expression of CGRP, and we found that repeated dural infusion of IS significantly increased the expression of CGRP(Fig. 1C). The above results show that we establish a successful and reliable rat model of CM, which can be used in follow-up experiments.
Upregulation of mGluR5 expression in the TNC of CM rats
After the successful establishment of the CM rat model, qRT-PCR and western blot analysis techniques were used to measure the expression of mGluR5 in TNC. The results of qRT-PCR showed that compared with the sham group, the mRNA level of mGluR5 in the CM group rats was significantly increased(Fig. 2A). Consistent with the results of qRT-PCR, the protein level of mGluR5 was significantly higher than that of the sham group(Fig. 2B). The results show that the expression of mGluR5 is upregulated in CM, which suggests that the mGluR5 signal may play an important role in CM.
Downregulation of mGluR5 attenuated the hyperalgesia and reduced the expression of CGRP in CM rats
We have confirmed that mGluR5 was significantly upregulated in CM rats. Therefore, we administered mGluR5 antagonist MPEP to the lateral ventricle to explore the effect of mGluR5 on hyperalgesia and CGRP. Compared with the sham group, the mechanical thresholds(Fig. 3A) and the thermal thresholds(Fig. 3B) of the CM group were significantly decreased. The administration of DMSO alone into the lateral ventricle did not affect the pain thresholds. The administration of Medium-dose(2 µg/5 µL) or high-dose(5 µg/5 µL) MPEP increased the mechanical pain thresholds and thermal pain thresholds of the CM rats, while the low-dose(0.2 µg/5 µL) had no obvious effect, so we choose medium-dose MPEP (2 µg/5 µL) for subsequent experiments. CGRP is expressed in trigeminal ganglion neurons and participates in the regulation of trigeminal nerve innervation and nociceptive transmission in CM. Consistent with the pain thresholds results, the western blot(Fig. 3C)and immunofluorescence(Fig. 3D) results showed that the administration of MPEP (2 µg/5 µL) reduced the high expression of CGRP in the CM rats. These results indicate that mGluR5 is involved in CM.
Downregulation of mGluR5 reduced expression of synaptic associated proteins
We detected synaptic associated proteins after administration of mGluR5 antagonist MPEP to explore whether mGluR5 can regulate the synaptic plasticity of CM. Western blotting was used to detect PSD and Syp, immunofluorescence was used to detect Syt-1. Compared with the sham group, the expression of PSD(Fig. 4A) and Syp(Fig. 4B) protein and the number of Syt-1-positive cells(Fig. 4C) in the CM group were significantly increased. There was no significant difference between the CM and CM + DMSO groups. The administration of MPEP decreased the expression of PSD and Syp proteins and the number of Syt-1-positive cells. These results suggest that mGluR5 may regulate the synaptic plasticity of CM.
MGluR5 regulated the ultrastructure of synapses
Synaptic structure is the structural basis of synaptic transmission and synaptic plasticity. We observed the synaptic structure of each group of TNC neurons through TEM(Fig. 5). In the sham group, the synapse structure was clear, the outline was complete, and many synaptic vesicles were seen. Compared with the sham group, the synaptic structure of the CM and the CM + DMSO groups was blurred, the synaptic cleft width, the thickness of PSD increased, and the synaptic interface curvature increased. The administration of MPEP restored the changes in the morphological indicators of synapses (Table 2).
MGluR5 regulated the PKC/NR2B signal in CM rats
To investigate whether mGluR5 regulates the synaptic plasticity through PKC/NR2B signal in CM, we detected the protein expression of PKC, total NR2B, and phosphorylation of NR2B at Tyr1472(pNR2B-Y1472). We found that the expression of PKC(Fig. 6A) and pNR2B-Y1472(Fig. 6C) significantly increased in the CM group, while the expression of NR2B(Fig. 6B) had no significant difference. After the lateral ventricle administration of MPEP, the PKC and pNR2B-Y1472 expression decreased. These results support the notion that mGluR5 may regulate synaptic plasticity by PKC/NR2B signal.
Downregulation of mGluR5 attenuated the central sensitization in CM rats
In order to explore whether mGluR5 is involved in the central sensitization of CM, we tested the expression of central sensitization-related indicators. CREB participates in the sensitization of noxious cells through phosphorylation. c-Fos is a classic marker of neuronal activation after noxious stimulation. We detected the expression of pCREB-S133 and c-Fos. The western blotting results showed that the expression levels of pCREB-S133(Fig. 7B) and c-Fos(Fig. 7C) in the CM group were significantly higher than those in the sham group, and DMSO alone had no obvious effect, and the administration of MPEP reduced the expression of c-Fos and pCREB-S133. The expression of CREB(Fig. 7A) had no significant difference. The results of c-Fos immunofluorescence(Fig. 7D) were consistent with the expression of the c-Fos protein. The above results indicate that mGluR5 may regulate the central sensitization of CM.