Animals

One hundred fifty-four male Sprague-Dawley rats weighing 200–300 g each were used in this study. They were housed in standard clear plastic cages with soft bedding, with access to food and water ad libitum. Studies were approved by the ethical committees of the Shanghai Ninth Hospital affiliated with Shanghai Jiaotong University. The guidelines for investigation of pain in animals given by the International Association for the Study of Pain were followed.17

Appliance for ETM in Rats

A fixed nickel-titanium alloy closed-coil spring appliance was connected between a maxillary first molar and the ipsilateral upper incisor, as described previously.18–20 After an intraperitoneal injection of sodium pentobarbital at a dose of 40 mg/kg body weight, 40 g of force19,20 was applied for 0 hours, 4 hours, 1 day, 3 days, 5 days, and 7 days. Sham-treated rats received the same procedures, but the springs in their mouths were not active. Forces of 10 g, 30 g and 50 g were applied respectively for 3 days.

Drug Application In Vivo and Behavioral Testing

Periodontal injection of exogenous recombinant CCL2 (10, 50, and 100 ng/µL, respectively; 3 µL in all, R&D Systems, United Kingdom) or 0.9% saline was done using a modified glass needle around the upper anterior incisors at six different sites (0.5 µL/site) in the sham-treated (spring inactive) rats under a short period of anesthesia induced by isoflurane.

To evaluate antagonist and the effects of increasing force, we chose day 3 as the appropriate time to assess behavior according to the change rhythms of CCL2 expression and nocifensive behavioral responses after ETM. The delivery of subarachnoid antagonists in the medullary region of rats was performed carefully on day 3 after ETM, according to procedures developed previously.21 The CCR2 antagonist (10 µL of a 10 nM solution; Calbiochem, Darmstatt, Germany) or CCL2 neutralizing antibody (100 ng/µL,22 10 µL, Millipore, Billerica, MA, USA) was applied under a short course of anesthesia.

The rats were videotaped 30 minutes after drug application to record behavioral responses characterized by directed face grooming, which has been validated as a reliable behavior for assessment of tooth movement pain in rats.19 According to the procedures developed in previous studies,19 the rats were videotaped for 10 minutes each time, starting at least 15 minutes after placement in the cage. Each animal was videotaped two times at 10-minute intervals.19 Videotaped behavior was analyzed offline by an investigator who was blinded to the information regarding the rats. Each rat was weighed on every interval day, and changes in body weight versus the previous weights were used for statistical analyses.

Immunofluorescence and Image Acquisition

The maxillary portion of the TG was selected for immunohistochemistry (IHC) study. Specimens were sectioned serially at a thickness of 8 µm with a cryostat along the axis. Three sections were selected randomly from among these serial sections in each specimen for IHC according to procedures described previously.20 Mouse anti-CCL2 (1∶100, Millipore) primary antibody was incubated overnight at 4°C. Hoechst was used for nuclear staining. Z-stacks of images taken at 1-µm intervals were acquired and photographed under the appropriate objective lenses of a Nikon A1 confocal microscope. To study the distribution of cell size, large (>40 µm), medium (30–40 µm), and small (<30 µm) neurons were carefully determined by scale bars, and the ratios of each type of neurons were calculated.

Western Blot

The corresponding maxillary portion of TG was collected for Western blot study according to the procedures described by previous studies.18 Mouse anti-CCL2 (1∶2000, Millipore) and goat anti-CCR2 (1∶1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were selected as primary antibodies. Actin was used as the internal control protein. Band intensity was assessed using Image Quant 5.2 software and protein levels were normalized to that of the 0-hour group in Figure 1, or 10-g group in Figure 3.

Trigeminal Neuron Cell Culture and Drug Treatment In Vitro

The TGs were removed from 2-month-old rats and cultured according to previous procedures.22 In brief, they were incubated for 1 hour in 0.15% collagenase (Worthington type I, Scima, Templestow, Victoria, Australia), followed by an additional 1 hour of incubation in 0.15% collagenase and 0.25% trypsin (Sigma, St. Louis, MO, USA). After 2 days of incubation, the neurons were used for the in vitro assay.

Recombinant rat CCL2 (R&D Systems, United Kingdom) was added to the culture plates for 15 minutes in the appropriate concentrations. Each antagonist was added 30 minutes before the addition of CCL2.

Statistics

For statistical analyses, one-way analysis of variance (least significant difference) and Student's t-test were used (SPSS 11.5, SPSS Inc, Chicago, IL, USA). The level of statistical significance was set at P < .05.

Time Course of the Changes in CCL2/CCR2 Expression in the TG

We observed a gradual increase in CCL2 and CCR2 expression that reached a statistically significant value after day 3, peaked on day 5 (Figure 1A), and down-regulated significantly after day 7 (Figure 1B,C). Also, the number of CCL2-immunoreactive (ir) neurons was statistically significantly increased on days 3 and 7 (Figure 1D,E). Further analysis showed that most of the ir cells were found in the small to medium-sized trigeminal neurons (Figure 1D,F), which are thought to play an important role in pain mechanisms.

Nociceptive Behavioral Responses Following ETM

ETM triggered a significant increase in nocifensive behavior on days 1, 3, 5, and 7. The amplitude of this nociceptive-like behavior seemed to reach a maximum on days 1 to 3 and decreased significantly on day 7 (Figure 2A).

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We further evaluated the changes in body weight after ETM as an indirect reflection of pain perception in the rats. As expected, body weight increased in the sham group and decreased in the experimental group until 5 days after ETM (Figure 2B).

The Effects of Increasing Force on the Nocifensive Behavioral Responses and CCL2 Expression in TG

To further validate our hypothesis, we tested the effect of increasing force on the changes in behaviors and CCL2 expression. As we expected, we found that both CCL2 expression intensity and positive cells in TG increased in a force magnitude–dependent manner (Figure 3A–D). Also, nocifensive grooming activities increased correspondingly in the same manner (Figure 3E).

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Effects of Exogenous Recombinant CCL2 and Its Antagonists on Nocifensive Behavioral Activities and C-Fos Expression

To illustrate CCL2's role in the trigeminal pain mechanism directly, exogenous CCL2 (3 µL of 10-, 50-, and 100-ng/µL solutions) was injected into periodontal tissues. This led to a significant increase in nociceptive-like behavioral responses in a dose-dependent manner (Figure 4A).

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In addition to the study of change in function, we also evaluated the pain behavioral responses after inhibiting CCL2/CCR2 signaling 3 days after ETM. Intrathecal application of CCR2 antagonist (10 µL of a 10 nM solution) or CCL2 neutralizing antibody (10 µL of a 100 ng/µL solution) decreased the nocifensive behavior significantly (Figure 4B).

We also applied exogenous CCL2 to cultured TG neurons in vitro. As expected, exogenous CCL2 led to a marked increase of c-fos expression within 15 minutes in a dose-dependent manner; this could be blocked by CCL2 neutralizing antibody (Figures 4C,D).