Background
Migraine is an incapacitating neurovascular disorder that substantially affects the quality of both life and work of patients. According to the number of headache days suffered per month, migraine can be classified as episodic migraine (EM) or chronic migraine (CM). The prevalence of CM is 1–3% in the general population and 2.5% of migraine patients develop CM each year, which seriously affects their life and work of patients [
1,
2]. In addition, the high medical expenses and the decreased ability of CM patients to work place an enormous financial burden on society. The severe headache and high morbidity in CM seriously harm patients’ physical and mental health. Therefore, the World Health Organization (WHO) has listed CM as one of the four most serious chronic dysfunction diseases [
3].
The physiopathology of CM is poorly understood. Most research conducted to date has suggest that activation of the trigeminovascular system (TGVS) contributes to the migraine development [
4,
5]. Hyperexcitability of neurons lead to neuropathic pain and trigger central sensitization. Central sensitization refers to increased synaptic efficacy in somatosensory neurons in the dorsal horn of the spinal cord following intense peripheral noxious stimuli, tissue injury or nerve damage. This heightened synaptic transmission can lead to a reduction in the pain threshold, the spread of pain sensitivity to non-injured areas and amplification of the pain responses [
6]. In chronic migraine, the release of calcitonin gene-related peptide (CGRP) and other excitatory neurotransmitters from the central terminals of trigeminal ganglion (TG) neurons could repetitively excite second-order neurons in the trigeminal nucleus caudalis (TNC), leading to central sensitization and the manifestation of hyperalgesia and allodynia [
7,
8]. Therefore, the neuron activation caused by enhanced synaptic transmission in central sensitization may plays a very important role in the maintenance of CM.
Additionally, most studies have suggested that the development and maintenance of central sensitization are largely dependent on the activation of the glutamate N-methyl-D-aspartate (NMDA) receptors [
9]. The combination of NMDA receptors and transmitters leads to an intracellular cascade that triggers a series of biochemical reactions resulting in alterations in the structure and function of the synapse, and these synaptic changes greatly enhance excitatory synaptic transmission and thus contribute to chronic pain [
10]. Some research on the brain has shown that the N-methyl D-aspartate receptor subtype 2B (NR2B) subunit is the most important tyrosine-phosphorylated protein, and the phosphorylation of NR2B receptor subunits has been proposed to lead to increased Ca2+ entry through the receptor in both central sensitization and NMDA-dependent synaptic plasticity [
11,
12]. However, the mechanism through which NR2B participates in CM-related central sensitization by altering synaptic plasticity has not been reported. We conducted a preliminary study and found that NR2B-pTyr might contribute to CM in rats, which manifested as decreased pain thresholds and exaggerated pain responses [
13]. Based on our previous studies, NR2B-pTyr was blocked by the administration of PP2 and genistein to investigate synaptic plasticity-related protein expression, the synapse ultrastructure, and the dendritic spine numbers and thus illustrate the resulting changes in the structural plasticity of the synapse. According to our data, NR2B-pTyr participates in the central sensitization-related mechanism of CM by regulating synaptic plasticity.
In this study, we aimed to explore the possible activity-dependent synaptic plasticity of NMDA receptors in CM, and our findings indicated that inhibition of NR2B-pTyr regulation of synaptic plasticity in central sensitization might be a novel and promising candidate for future treatment or prevention of CM.
Methods
Animals
Total up to 149 male adult Sprague-Dawley rats (250–300 g) were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The experimental groups are shown in Table
1. Rats were allowed free access to water and food and were housed at 23 ± 1 °C under a 12/12 h light-dark cycle. Before any experimental procedures, all animals were acclimated for at least 7 days. All the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1996). Because this model induces pain in animals, the number of rats used was the minimum necessary to achieve a sufficient level of power for the statistical power.
Table 1
Animal numbers in each group
sham | 10a | 6 | 6 | 6 | 6 | | 24 |
CM | 10a | 6 | 6 | 6 | 6 | 2 | 26 |
CM + DMSO | 10a | 6 | 6 | 6 | 6 | 2 | 26 |
CM + PP2 (7.3 nmol) | 10 | | | | | 1 | 11 |
CM + PP2 (73 nmol) | 10a | 6 | 6 | 6 | 6 | 1 | 25 |
CM + genistein (100 ng/g) | 10 | | | | | 1 | 11 |
CM + genistein (300 ng/g) | 10a | 6 | 6 | 6 | 6 | 2 | 26 |
Total | 20 | 30 | 30 | 30 | 30 | 8 | 149 |
Craniotomy and cannula fixation
Rats were fitted with a cranial chamber, deeply anaesthetized with 10% chloral hydrate (i.p., 0.4 g/kg body weight), and placed in a stereotaxic apparatus (ST-51603; Stoelting Co, Chicago, IL, USA). Following disinfection with iodophor and alcohol, an incision was made along the midline of each rat’s head to fully expose the skull. A skull drill was used to perform a 1-mm-diameter craniotomy in the right frontal bone (+ 1.5 mm from the bregma and + 1.5 mm lateral to the bregma), and a sterile stainless-steel cannula with a plastic cup (RWD, Shenzhen, China) was affixed to the bone using dental cement. The end of the cannula opened onto the dura, allowing inflammatory soup (IS) or phosphate-buffered saline (PBS) to contact the dura. A matched obturator cap was used to seal the cannula. After surgery, antibiotics were topically applied to prevent any infections in the operation region. The rats were then maintained at approximately 37 °C on an electric heating blanket and housed separately until complete recovery from anaesthesia. The rats were allowed recover for at least 7 days prior to dural infusions. All the rat experiments were approved by the Ethics Committee of the Department of First Affiliated Hospital of Chongqing Medical University Medical Research.
Repeated dura infusions
A CM rat model was established by repeated infusions of IS to the dura in conscious rats. We modeled recurrent trigeminovascular or dural nociceptor activation that is assumed to occur in patients with CM, as described previously [
14]. Rats were placed in a box that allowed free movement for the infusion of IS or PBS to the dura. The IS contained 1 mM histamine, 1 mM serotonin, 1 mM bradykinin, and 0.1 mM prostaglandin E2 in PBS (pH 7.4). What is said above chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). We provided a steady infusion of 2 μl of IS or PBS was provided through the cannula for 10 min while each rat was freely moving. The tube was left in place for at least 5 min after the infusion to allow the IS or PBS to diffuse into the tissue surrounding the dura, and the cap was returned to the cannula after the infusion. In addition, we inspected the skin and dental cement seal around the cap to ensure no leakage of IS or PBS outside the dura onto the skin. The rats were randomly divided into two groups and infused with IS or PBS for 7 days.
Animal groups and treatment
Animals were randomly divided into the following groups: the (1) sham group, (2) CM group, (3) CM + dimethyl sulphoxide (DMSO) group, (4) CM + PP2 group, and (5) CM + genistein group. The animals in the sham group were slowly infused with 2 μL of PBS (pH 7.4) into the dura, as described above, whereas those in the CM group were infused with 2 μL of IS. To investigate the role of NR2B-pTyr in the intracellular events after CM, we dissolved the NR2B-pTyr inhibitor PP2 (Abcam, USA) or genistein (Beyotime, China) in DMSO and injected it into the lateral ventricle (− 1.0 mm rear from the rear of the bregma, + 1.5 mm lateral to the bregma, 4.0 mm from the skull plane) with the designated treatment solution (5 μL). An equivalent volume of DMSO was injected into the lateral ventricle as a control. The doses of PP2 (7.3 and 73 nmol) and genistein (100 and 300 ng) used in this study were based on previous studies [
13,
15].
Tactile sensory testing
As previously described, we used the von Frey test to detect the mechanical threshold in the periorbital and hind paw regions. Mechanical thresholds were tested before the first IS or PBS infusion to serve as the baseline. In addition, the test was performed 24 h after each dural infusion and before the next IS or PBS stimulation (n = 10, each group). Tactile sensory testing was performed 24 h after the administration of PP2, genistein or DMSO (n = 10, each group) to determine these infusions on mechanical thresholds. Briefly, the rats were placed in the testing apparatus and were acclimated to the testing apparatus during training periods before and after the cannula implantation surgery. Pressure thresholds were determined by applying an electronic von Frey device (Electrovonfrey, model no: 2391, IITC Inc., Woodland Hills, CA, USA), and the assigned force values ranged from 0 to 800 g. According to the manufacturer’s instructions, the pressure probe tip was applied to the periorbital region and hindpaw region of the rats, and the threshold was automatically recorded when the rat quickly retracted its head or hind paw away from the rigid tip. The results for the PBS group were considered to indicate the control mechanical threshold. Threshold values were measured at least three times at each site, with an interval of at least 1 min between tests. The results are presented as the thresholds in g ± standard deviations (SDs). The data were recorded separately for each time point.
Western blot analysis
We examined the expression of total NR2B (tNR2B), pNR2B-Y1472, pNR2B-Y1252, PSD95, Syp, Syt-1, and CGRP through a western blot assay (n = 6 in each group). Twenty-four hours after the administration of PP2 and genistein, the rats were euthanized, and the brains were removed. The TNC tissue was then separated. Cut TNC tissues into pieces and homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (sc-24,948, Beyotime, China) with protease inhibitor (Beyotime, China) and phosphatase inhibitor (Beyotime, China) at 4 °C for 2 h. The homogenate was centrifuged at 14,000 rpm at 4 °C for 20 min, and the protein concentrations were then determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, China). The supernatant was used as a whole-cell protein extract. Equal amounts of protein were loaded onto a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime, China), electrophoresed, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked with 5% nonfat milk at 37 °C for 2 h and incubated with primary antibodies, including anti-NR2B (1:1000, Proteintech), anti-NR2B phospho Y1252 (1:500, Abcam), anti-NR2B phospho Y1472 (1:500, Abcam), anti-PSD95 (1:1000, Abcam), anti-synaptophysin (1:5000, Abcam), anti-synaptotagmin1 (1:500, Bioss), anti-CGRP (1:2000, Abcam), and anti-β-actin (1:5000, Proteintech, USA) at 4 °C overnight. The membranes were washed with Tris-buffered saline Tween 20 (TBST) buffer three times and incubated with a secondary antibody (1:5000, Zhongshan Golden Bridge Bio, China) at 37 °C for 2 h. The immunoblots were probed with a western blot detection kit (Advansta, USA) and visualized with an imaging system (Fusion, Germany). β-actin was used as a loading control to normalise the protein levels.
Immunofluorescence staining
Rats were anaesthetized and transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS 1 day after the administration of PP2 or genistein. Regions from the medulla oblongata to the first cervical cord were separated immediately, post-fixed in 4% paraformaldehyde for 24 h, and then sequentially immersed in solutions of sucrose with increasing concentrations (20% to 30%) until the tissue sank to the bottom. Segments of the TNC were cut into 10-μm-thick sections with a cryostat (Leica, Japan). All the sections were stored at − 80 °C for later use. For immunofluorescence staining, the sections were washed three times with PBS and permeabilised with 0.3% Triton X-100 (Beyotime, China) in 0.1 M PBS at 37 °C for 10 min and then incubated with 10% normal goat serum (Boster, China) at 37 °C for 30 min, using a neuronal marker (anti-neuronal nuclei (NeuN), mouse, 1:200, Novus), anti-PSD95 (1:500, rabbit, Abcam), anti-synaptophysin (1:500, rabbit, Abcam), anti-synaptotagmin1 (1:200, rabbit, Bioss), anti-CGRP (1:50, mouse, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-SP (1:50, mouse, Abcam) at 4 °C overnight. Then, after three washes with PBS, the sections were incubated with secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG, 1:200, Beyotime, China), Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200, Beyotime, China), and Cy3-conjugated goat anti-mouse IgG (1:200, Beyotime, China) at 37 °C for 90 min. Microphotographs were obtained analysed with a fluorescent confocal microscope (ZEISS, Germany). PBS rather than primary antibody was applied to the negative control sections, and no positive signals were detected. The expression levels of CGRP, SP, PSD95, Syp and Syt-1 in the TNC were detected by immunofluorescence staining (n = 6 in each group, five images per animal), and five sections from each rat were randomly selected. A 20x objective was used to capture PSD95- and Syt-1-immunoreactive cells and the intensity of Syp immunoreactivity. A 10x objective was used to capture bilateral CGRP and SP immunoreactivity in the TNC, and CGRP and SP expression did not differ between the two sides. The number of positive cells was calculated as the mean of the numbers obtained from five images.
Transmission electron microscopy
Six rats per group were anaesthetized and perfused with 2.5% glutaraldehyde, and their brains were dissected and removed. The TNC was separated and incubated overnight in 4% glutaraldehyde at 4 °C for 24 h. Then, the TNC was cut into 1-mm
3 pieces with a blade. Post fixing, embedding, sectioning and staining were performed at Chongqing Medical University. Briefly, the 1-mm
3 tissue blocks from the TNC were washed three times with PBS and fixed in 1% osmium tetroxide for two hrs. In addition, the tissue blocks were dehydrated in a series of graded aqueous ethanol for 10 min each (50%/70%/90%/2 × 100%). In addition, the tissue blocks were transferred to 100% propylene oxide for 15 min, followed by graded resin infiltration and embedding. Ultrathin sections were prepared on a Leica Ultracut T using a 45-degree diamond histoknife. The tissue was washed two times with distilled water and stained en bloc with 2% uranyl acetate and lead citrate for 45 min. Images were taken using a JEM-1400 PLUS transmission electron microscope (TEM) and analysed using Image Pro Plus. Synaptic morphology parameters were measured at 50000x. The width of the synaptic cleft and the thickness of the postsynaptic density (PSD) were measured using a multi-point averaging method and Guldner’s [
16] method. The synaptic interface curvature was obtained using Jones’ [
17] method (
n = 6 in each group, five images per animal).
Golgi-cox staining
One day after the administration of PP2 or genistein, the rats were injected intraperitoneally with a lethal dose of chloral hydrate to induce anaesthestia (
n = 6 in each group, five images per animal). The brains were removed as soon as possible without perfusion, and the tissue was rinsed in double-distilled water for 2–3 s to remove blood from the surface. An FD Rapid Golgi Stain Kit™ (FD NeuroTechnologies—Columbia, MD, USA) was used for the tissue preparation and staining procedure. The entire Golgi-Cox staining procedure was conducted in strict accordance with the manufacturer’s user manual and material safety datasheet. The extracted brains were immersed in Rapid Golgi-Cox solution (“Solutions A/B”) for 14 days (the solution was changed once after 24 h) at room temperature (RT) with low ambient light, transferred into cutting solution (“Solution C”), sectioned on a vibratome (Leica VT 1200S, Japan) at 200 μm to ensure that whole (untransected) neuronal arbours could be accommodated and then mounted on gelatine-coated slides. The slides were further developed and processed according to the manufacturer’s instructions and then coverslipped with Permount™ Mounting Medium (Fisher Scientific Co, Waltham, MA, USA). Briefly, the dendrites within the region were imaged with a Zeiss microscope (Axio Imager A2) using 40x and 64x objectives, and the dendritic spines were quantified by an experimenter who was blinded to the group of each sample [
18].
Statistical analysis
The data are expressed as the means ± SDs. Statistical analyses were performed with SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and graphs were generated by GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). The mechanical thresholds of the sham and CM groups were assessed using two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. One-way ANOVA followed by a Bonferroni post hoc test was used to compare the differences among multiple groups. Statistical differences between two groups were analysed using independent-sample t-tests. P < 0.05 was considered to indicate statistical significance.
Discussion
In the present study, we used the von Frey test to detect hyperalgesia and allodynia and thus investigate the role of NR2B and NR2B-pTyr in rats with CM induced by repeated infusions of IS. In addition, the use of PP2/genistein to suppress the tyrosine phosphorylation of NR2B ameliorated the hyperalgesia induced by repeated IS stimulation and downregulated the expression of CGRP and SP. Moreover, the inhibition of NR2B-pTyr by PP2/genistein downregulated the expression of the synapse-associated proteins PSD95, Syp, and Syt-1 and even altered the synaptic ultrastructure and the number of dendritic spines to reduce synaptic plasticity. Based on these results, we provide the first evidence showing that NR2B-pTyr in the TNC is involved in the mechanism of CM. The effect of NR2B-pTry was found to be largely associated with its regulation of synaptic plasticity. Therefore, the tyrosine phosphorylation of NR2B and synaptic plasticity might represent potential therapeutic targets in CM.
Central sensitization is a crucial process underlying increased neuronal excitability, and some published evidence indicates that central sensitization plays a role in the maintenance of prolonged migraine pain and might also contribute to migraine chronicity [
6,
22,
23]. Repeated dural nociceptor activation specifically leads to gradual worsening of cutaneous hypersensitivity, general neuronal hyperexcitability, persistent cephalic cutaneous hypersensitivity and trigeminal central sensitization [
24]. In the present study, we used a CM rat model established by repeated IS stimulation of the dura to repeatedly activate the assumed nociceptors, We discovered that the periorbital and hind paw pain thresholds gradually declined with repeated infusions of IS. This finding suggests that IS induces cephalic and extracephalic allodynia.
NMDA receptors constitute one of the principal types of ionotropic glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Abundant evidence indicates that NMDA receptors play critical roles in a range of physiological and pathological processes in the CNS. One of the key mechanisms for regulating NMDA receptor function involves the tyrosine phosphorylation of NR2B subunits by the tyrosine kinase Fyn [
25,
26]. NR2B contains three tyrosine phosphorylation sites (Y1252, Y1336 and Y1472), and several studies have suggested that pNR2B-Y1472 plays a significant role in the trafficking of NMDA receptors [
25,
27,
28]. In an animal model of inflammatory pain, the development of inflammation and hyperalgesia was found to be associated with a rapid and prolonged increase in the pNR2B-Y1472 level. In addition, the inflammation-induced increase in NR2B-pTyr was abolished by genistein, a tyrosine kinase inhibitor, and PP2, a Src family protein tyrosine kinase inhibitor [
29]. In agreement with previous studies, we found that pNR2B-Y1472 was involved in IS-induced hyperalgesia. The protein levels of pNR2B-Y1472 were overexpressed in the TNC of CM rats, and only high doses of PP2 (73 nmol) and genistein (300 ng) relieved mechanical hyperalgesia. In addition, we found the same changes in the phosphorylation of Y1252 sites. However, other remaining phosphorylation sites should be further investigated. These results suggest that the phosphorylation of NR2B at Y1472 and Y1252 regulated CM activation in TNC neurons.
CGRP and SP are important in migraine pathophysiology, expressed in trigeminal ganglia neurons and involved in trigeminovascular innervation, and modulation of nociceptive transmission. Additionally, these proteins are used as biological markers of neuronal activation and central sensitization [
20,
30]. As shown in previous studies, the plasma CGRP and SP levels are increased during a migraine attack [
31,
32]. In our study, upregulated expression levels of CGRP and SP were observed in the TNC of CM rats, and CGRP and SP immunoreactivity was mainly detected in the outer laminae of the TNC, which is likely associated with the processing of nociceptive information. This finding is in line with the results of a previous study [
30]. In addition, PP2 and genistein downregulated the levels of CGRP and SP in the TNC, which indicates that NR2B phosphorylation might play a prominent role in neuronal activation and central sensitization in the CM model.
In addition, NR2B-pTyr was reported contributes to the development of persistent pain in the spinal cord by regulate synaptic transmission. Therefore, It’s very interesting to explore the mechanism of NR2B-pTyr contributes to central sensitization in CM. As is well known, excitatory neurotransmission in somatosensory nociceptive pathways is predominantly mediated by glutamatergic synapses [
33]. Recent studies have consistently demonstrated that glutamatergic synapses play an important role in sensory transmission, including pain and itch transmission, and contribute to nociceptive sensitization [
34,
35]. Many studies have reported that regulation of synaptic plasticity by NR2B-pTyr may play a role in nociceptive transmission in the chronic visceral pain model and neuropathic pain model [
36,
37]. Similar to a previous report, we detected many synaptic-related indicators in the CM model to emphasize that its association with the synaptic plasticity of NMDA receptors. PSD95 is preferentially localized to dendritic spines and plays a critical role in the regulation of the size and shape of dendritic spines [
38,
39]. Syp, a major integral membrane protein of presynaptic vesicles, is required for vesicle formation and exocytosis and is widely used as a marker for synaptic activity [
40]. Syt-1, which acts as the major Ca2 + −sensor for fast presynaptic vesicle exocytosis, is a marker of synaptic transmission. Similar to previous studies, our study revealed that the expression levels of synaptic function/structure-related proteins PSD95, Syp and Syt-1 were increased in CM rats and that PP2 and genistein downregulated these changes. This finding suggests that the expression of synaptic function/structure-related proteins in the TNC is directly related to CM rats and positively correlated with the level of NR2B-pTyr. However, electrophysiological experiments are necessary to more fully describe these changes in synaptic plasticity changes.