Microglia P2X4 receptor contributes to central sensitization following recurrent nitroglycerin stimulation

Male C57BL/6 mice (weighting 18–20 g, aged 8–10 weeks) were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). All procedures followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Mice were maintained on a 12-h light/dark cycle under standard laboratory conditions with enough food and water. Experiments were carried out between 9:00 and 15:00. All animals were acclimatized for 1 week before experimental procedures and randomly assigned to experimental groups. Randomized allocation was designed with Excel Software. The procedure was as follows: (1) C67BL/6 mice weighting 18–20 g were selected then numbered. (2) The randomization numbers were created using the “rand()” function of Excel software and sorted by incremental method. (3) Then, according to the order, each 6–8 numbers are divided into different groups.

NTG (Beijing Regent, China) was prepared from a stock solution of 5.0 mg/ml dissolved in 30% alcohol, 30% propylene glycol, and water. NTG was diluted at 1 mg/ml prior to the start of the experiment. The vehicle control was 0.9% saline because there was no significant difference in mechanical thresholds between 0.9% saline and the solution in which NTG was dissolved (6% propylene glycol, 6% alcohol, 0.9% saline). Animals received intraperitoneal (i.p.) injections of 10 mg/kg NTG or vehicle every other day for 9 days (i.e., days 1, 3, 5, 7, and 9) [13].

To understand the effect of microglia and P2X4R on NTG-induced hyperalgesia, we treated the animals with the microglia inhibitor minocycline (MedChemExpress/MCE, American) or the P2X4R-selective antagonist 5-BDBD (Sigma-Aldrich, American). Minocycline and 5-BDBD were dissolved in DMSO solution, which was used as the vehicle control. Minocycline/vehicle or 5-BDBD/vehicle was administered immediately prior to the administration of NTG/saline [14, 15]. The treatment of the mice on testing days was as follows: mice were habituated to the test room, followed by a baseline measurement of mechanical thresholds 15–20 min later and an administration of minocycline (30 mg/kg, i.p.), 5-BDBD (28 mg/kg, i.p.), or vehicle; then, an injection of NTG/saline was given, and 2 h later, acute mechanical responses were tested. This procedure was repeated every other day for 9 days.

All animal behavior testing occurred in light conditions, between 09:00 and 15:00. For the determination of mechanical sensitivity, the threshold for responses to punctuate mechanical stimuli (mechanical hyperalgesia) was tested according to the up-and-down method. The plantar surface of the animal hind paw was stimulated with a series of eight von Frey filaments (bending force ranging from 0.01 to 2 g) from the underside of the mesh stand. The 0.4-g von Frey filament was tested first. A response was defined as a withdrawal, shaking, or licking of the paw. In the absence of a response, a heavier filament (up) was used, and in the presence of a response, a lighter filament (down) was tested. Each filament was applied to the skin for 3 s at 1-min intervals. This pattern was followed for a maximum of four filaments following the first response. The evaluating experimenters were blind to the experimental groups.

Mice were euthanized, and the TNC was dissected at 12 h after last NTG injection for qRT-PCR experiments. The tissues were immediately stored in liquid nitrogen. Total RNA was extracted as previously described [16] using an RNAiso Plus reagent (TaKaRa, Dalian), and RNA was quantified spectrophotometrically with NanoDrop (Thermo, USA). Then, cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara). P2X4R (p2rx4) mRNA expression are analyzed. Specific primers were obtained from Sangon Biotech (Shanghai, China). The sequences of primers are described as follows: P2X4: 5′-TCG TGT GGG AAA AGG GCT AC-3′ (forward), 5′-GTC TGG TTC ACG GTG ACG AT-3′ (reverse); GAPDH: 5′-ATG ACT CTA CCC ACG GCA AGC T-3′ (forward), 5′-GGA TGC AGG GAT GAT GTT CT-3′ (reverse). GAPDH mRNA was used as an endogenous control for normalization. Quantitative real-time PCR was performed with SYBR® Premix Ex Taq™ II (Takara) with a CFX96 Touch thermocycler (Bio-Rad, USA) according to the instructions. All fluorescence data were processed by a post-PCR data analysis software program. The ΔΔCq method was used to investigate the differences in the gene expression levels.

For P2X4R protein detection, TNC was collected 12 h after different NTG injection times. Until measurements, the samples were stored at − 80 °C. Tissues were sonicated in an ice-cold RIPA lysis buffer (Beyotime, China) containing with phenylmethylsulphonyl fluoride (PMSF, Beyotime, China) at 4 °C for 1 h. Protein concentration was measured according to the BCA protein assay method with a BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein (40 μg) were loaded on an SDS-PAGE gel (Beyotime, China), electrophoresed, and transferred to PDVF membranes. Following the transfer, membranes were blocked for 2 h at room temperature in TBST containing 5% non-fat milk and incubated in a primary antibody, rabbit anti-mouse P2X4R polyclonal antibody (1:1000, Abcam). The complete information of all antibodies were included in the Additional file. The next day, membranes were incubated in a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:9000, ZSGB-BIO, China) at 37 °C for 1 h. Immunoblots were then probed with a BeyoECL Plus kit (Beyotime, China) and visualized with an imaging system (Fusion, Germany). β-Actin (1:9000, Proteintech, China) was used as a loading control to normalize protein levels. 1

For c-Fos detection, mice were treated as described above with 5-BDBD or vehicle, and tissue was collected 2 h after the last NTG injection. Mice were anesthetized with 10% chloral hydrate (0.1 ml/10 g) and perfused intracardially with 30 ml of ice-cold 0.9% saline and subsequently perfused with 50 ml ice-cold 4% paraformaldehyde (PFA)/0.1 M PBS (pH 7.4). The whole brain was collected and post-fixed in 4% PFA at 4 °C for 18 h. The medullary segment containing the TNC between + 1 and − 3 mm from the obex was removed. Tissues were immersed in 20% and 30% sucrose in turn. Samples were then frozen, and transverse sections were cut at 10 μm thickness (Leica). For c-Fos staining, sections were incubated in 0.3% H₂O₂ for 30 min and blocked with 10% normal goat serum in PBST (10% NRST) for 1 h, followed by incubation with anti-c-Fos antibody at 4 °C (1:500, 48 h, Abcam, USA). Sections were incubated for 1 h with a goat anti-rabbit secondary antibody and incubated with an avidin-biotin-complex using a kit (ZSGB-BIO, China). C-Fos primary antibody staining was visualized with DAB.

The TNC region was determined based on morphological appearance under light microscopy using the Mouse Brain Atlas as a reference [17]. Images were observed in a ZEISS Axio Imager A2 microscope under × 10 and × 20 objective lens. Expression of c-Fos was quantified by averaging 6–10 sections containing both sides of the TNC (throughout rostrocaudal extent) per mouse, (n = 7 mice/group). Image analysis was performed by Imagepro-Plus 6.0 software. The c-Fos-positive cells were scored as those with obvious specific nuclear staining. The laminae I–V of TNC were determined manually as area of interest using a rectangular tool. There was no significant difference in the area of TNC across experimental groups (see Additional file 2). All evaluations were performed by an observer blind to the experimental groups.

For Iba-1 and P2X4R detection, tissues were collected at 12 h after the last NTG/saline injection. But considering the short half-life of CGRP, tissues were taken at 2 h after last NTG/saline injection for CGRP immunofluorescence staining. The sections were blocked by incubation in 10% normal donkey serum or goat serum at room temperature for 30 min. Sections were incubated with goat anti-mouse Iba-1 polyclonal antibody (1:400, Abcam), rabbit anti-mouse P2X4R antibody (1:80, Abcam), or mouse anti-mouse CGRP antibody (1:100, Santa Cruze) overnight in 4 °C, respectively. The next day, sections were incubated with species-specific fluorophore-labeled secondary antibodies (1:400, Abbkine) for 2 h at room temperature. Nuclei were counter stained with 4′,6-diamidino-2-phenylinodole (DAPI) at 37 °C for 5 min. Sections were viewed under a confocal laser scanning fluorescence microscope (ZEISS, Germany).

The TNC area on the section images was determined as previously described. Subsequently, we calculated the ratio of cross-sectional area immunoreactive for Iba1 in the TNC area and estimated the number of Iba1-IR cells per 1 mm² of the TNC at × 10 or × 20 magnification using an image analysis software (Image-Pro Plus 6.2, Media Cybernetics). Optical density (OD) values were taken to indicate the quantities of CGRP and calculated by correcting the intensity of stained areas for the intensity of the background. The two sides of the TNC from six sections per animal (n = 6 mice/group) were included in the analysis.

Data are expressed as mean ± SD. All statistical analyses were performed by GraphPad Prism version 6.0 (Graph Pad Software Inc., San Diego, CA). All data were tested for normality using the Kolmogorov-Smirnov (K-S) normality tests and considered normally distributed data. F test or Bartlett’s test were used to compare variances. Behavioral results were analyzed using a two-way RM ANOVA, with drug and time as factors. In this case, all groups were compared to responses on day 1, and to the VEH-VEH group. For Iba-1 immunofluorescence and P2X4R qRT-PCR experiments, data were analyzed using unpaired t tests. If the variances were significantly different, unpaired t test with Welch’s correction was used. The expression of P2X4R and CGRP and the number of c-Fos-positive nuclei were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test, and the variance difference was not significant. A significance level of p < 0.05 was used.

Compared with the NTG group (NTG), minocycline (NTG + Mino) reduced the number of Iba1-labeled cells on the ninth day (532.9 ± 34.6/mm² vs 146.5 ± 16.7/mm², p < 0.05). Meanwhile, minocycline also reduced the ratio of the cross-sectional area of Iba1-IR cells to the TNC area (1.49 ± 0.2% vs 0.44 ± 0.1%, p < 0.05) (Fig. 2c–e). These results indicated that migraine-associated pain evoked by NTG may depend on microglia activation.

We used chronic intermittent NTG injection (10 mg/kg, i.p.) to build a CM animal model, described recently by Pradhan et al. [13]. NTG is a widely used migraine trigger in human clinical experiments [20]. Acute NTG-induced thermal and mechanical hyperalgesia in rodents has been used as an animal model for migraine. Moreover, chronic administration of NTG to mice resulted in not only acute mechanical hyperalgesia after each exposure but also sustained and progressive decrease in the basal mechanical threshold of hind paws. These results are consistent with the clinical observations of CM patients in whom hyperalgesia or allodynia may occur in both cephalic region and extracephalic region, such as the neck and forearms. The appearance of cutaneous allodynia following NTG likely reflects the development of central sensitization, which may be mediated through abnormal neuronal excitability within the trigeminal nucleus caudalis (TNC) and thalamus. Some researches determined the mechanical sensitivity of periorbital region and hind paws. Similar to the hind paws, NTG treatment also induces the periorbital hyperalgesia [21]. So in our study, we only tested the mechanical sensitivity of animal hind paws. This chronic basal hyperalgesia persists for days following cessation of NTG administration. Further, repeated administration of NTG has also been shown to produce photophobia, hypoactivity, and facial grimace behaviors [22]. Therefore, this is a reliable animal model for studying chronic migraine-associated pain.

In our study, recurrent NTG administration produced a significant basal sensitization, which was first observed after two NTG injections (i.e., day 5). In addition, on each test day, NTG also evoked significant acute mechanical hyperalgesia.

Most research on central sensitization mainly focuses on neuronal function, ignoring the potential contribution of gliocytes. Extensive studies about animal models of neuropathic pain have indicated that spinal glial activation, especially microglia, and the subsequent secretion of mediators have an important role in initiating or maintaining an aberrant hyperalgesia state [23].

Recently, some evidences have indicated that altered microglia function may be involved in the pathophysiological mechanisms of migraine. It is known that recurrent CSD is the upstream neural signal change in CM [24]. A recent study indicated that multiple CSD caused a marked enlargement of microglia [5] and release of microglia cytokine, such as IL-1β and TNF-α. These cytokines also affected the threshold and amplitude of CSD [25–27]. In addition, Cui Y and colleagues also found in a PET study that microglia were activated in the rat brain as a consequence of CSD stimulation [28].

In addition, Nathan Fried used another CM animal model that was built with repeated IS stimulation of dura mater. Then, the author indicated that no microglia activation was observed during the episodic stage (only two IS stimulations), but microglia activation was increased in the TNC during the chronic stage (total of ten IS stimulations). Importantly, minocycline treatment could inhibit trigeminal sensitivity after recurrent IS stimulation [7].

Our results showed that after recurrent NTG injection, the number of Iba1-IR cells was increased in the TNC. Minocycline significantly attenuated the NTG-induced basal mechanical hyperalgesia and immunoreactive staining for Iba1, but it did not alter acute hyperalgesia. Thus, inhibition of microglial activation can be used as a potential preventive therapy rather than an acute analgesic. However, in addition to its effects on microglia, minocycline has been shown to have broad effects on immune cells, such as astrocytes, oligodendrocytes, and macrophages. As such, data generated with minocycline need to be interpreted with caution. We cannot rule out that minocycline may affect other immune cells to decrease NTG-induced hypersensitivity. In addition, the cellular mechanism that underlies the crosstalk between microglia and neurons of the TNC needs further study.

Although N-methyl-D-aspartate (NMDA) and non-NMDA receptors are thought to be fundamental mechanisms underlying central sensitization, many studies suggest that purinergic receptor may be involved [29]. In the purinergic hypothesis for migraine, ATP has been implicated in migraine via stimulating cerebral vasodilatation and primary afferent nerve terminals located in the cerebral microvasculature.

A substantial number of studies have revealed an increasing microglia-specific P2X4R expression in the spinal cord in diverse animal models of neuropathic pain. In our experiments, a similar change in P2X4R expression in the TNC was found. Furthermore, the P2X4R-specific inhibitor, 5-BDBD, inhibits P2X4R-mediated Ca2+ signals and inward ion currents in vitro and is known to permeate the blood-brain barrier [30]. We found that 5-BDBD, unlike minocycline, effectively prevented both basal hyperalgesia and acute NTG-induced hyperalgesia. P2X4R is also expressed in other areas of the brain; therefore, we cannot exclude the possibility that 5-BDBD acts on other migraine-related regions including the periaqueductal gray (PAG), sensory cortical region, or even satellite glial cells (SGCs) of the trigeminal ganglion (TG) [31]. In addition, minocycline does not affect the activation of SGCs in the TG [32, 33]. Therefore, minocycline and 5-BDBD may play different roles in NTG-associated hyperalgesia.

C-Fos, the protein of the protooncogene c-fos, has been widely used as a marker for neuronal activation. Most treatments inhibiting c-Fos similarly affect pain-related behaviors. In addition, c-Fos has been implicated in central sensitization via transcriptional regulation of prodynorphin [34]. Studies using different animal models of migraine (including injection of NTG, IS, or CSD stimulation) all evoked an increase expression of c-Fos within the TNC, especially in laminae I and II [35]. In our study, a chronic NTG model significantly induced c-Fos expression in the superficial lamina of the TNC, where the first-order trigeminal neurons make synaptic contact with the second-order neurons. In addition, 5-BDBD alleviated NTG-induced c-Fos expression, which may underlie the reduction in the pain threshold.

CGRP, as a key neuropeptide of the trigeminal system, is implicated in the peripheral and central sensitization of migraine. During migraine attacks, the levels of CGRP in patient serum, saliva, and cerebrospinal fluid are elevated. In the animal model of migraine, NTG injection results in an increase of CGRP in the blood, dura mater, TG, and TNC [36]. However, some previous studies found that NTG treatment induced a decrease in the area occupied by CGRP-immunoreactive fibers and in CGRP protein expression by western blot in the TNC [37, 38]. These results indicated that this is a probably consequence of an increase release of CGRP from storage vesicles in the central terminals of trigeminal afferents. The majority of CGRP is synthesized in cell bodies of TG. Then, CGRP protein is transported in vesicles along the nerve fibers to the nerve endings in the TNC or solitary tract nucleus [39]. In our study, 2 h after NTG treatment was the time of sampling for determining the CGRP protein levels; these periods might be insufficient for the depleted CGRP to be resynthesized and to be transported to TNC. So we found a reduction of CGRP staining after repeated NTG injection. In addition, NO synthesis or cGMP activation may be involved in the mechanism of NTG-induced CGRP release [40].

In our studies, CGRP-immunoreactive fibers were mainly distributed in the superficial of TNC, which are associated primarily with processing nociceptive information. 5-BDBD pretreatment mitigated the NTG-induced CGRP immunoreactivity changes in the TNC, modulating the trigeminal system activation. The cellular mechanism underlying the P2X4R-induced CGRP release may be related to brain-derived neurotrophic factor (BDNF). Previous studies demonstrated that BDNF is released from ATP-stimulated microglia via the P2X4R signaling pathway in vitro and vivo [41]. In addition, BDNF, acting via trkB receptors, can regulate the expression and release of CGRP from sensory neurons [42].

Additional file 1:Complete information for antibodies in the research. (DOCX 16 kb)Additional file 2:Figure S1. Western blot for CGRP in the vehicle group on day 1. Figure S2. Western Blot for P2X4R in the vehicle group on day 1. Figure S3. There was no difference in the size of TNC areas. (DOCX 6150 kb)