Analgesic effect of gastrin-releasing peptide in the dorsal horn

Animals

Adult male Sprague–Dawley rats weighing 250–300 g were purchased from Japan SLC, Inc and used in all experiments in this study. The animals were maintained in a 12-h light/dark cycle with light on at 8:00 a.m. in a temperature-controlled (25.0°C±1.0°C) room, were group-housed in cages (two animals per cage), and had free access to food and water. Rats were anesthetized with sevoflurane (3.0%–5.0% inhalation administration, in air) during all operations.

All animal experimental procedures were approved by the Hyogo College of Medicine Committee on Animal Research (approval number, 20–024) and were conducted in accordance with the National Institutes of Health guidelines on animal care. All efforts were made to minimize suffering.

Animal model

We used formalin and Complete Freund’s adjuvant (CFA) for the acute and inflammatory pain models, respectively. Rats were anesthetized with sevoflurane (3.0%–5.0% inhalation administration, in air), and formalin (50 μL of a 1.5% solution of formaldehyde with saline) or CFA (100 μL of a 1:1 mixture of saline/CFA) was injected into the plantar surface of the left hind paw using a 1-mL tuberculin syringe equipped with a 30-gauge needle.

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) was performed as previously described. ¹⁶ Briefly, 2 h after formalin administration, rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 300 mL of 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4), followed by 500 mL of 4% PFA in 0.1 M PB.

The spinal cords were dissected out and post-fixed in the same fixative at 4°C overnight, followed by immersion in 20% sucrose in 0.1 M PB at 4°C for 3 days. The bilateral L4–L5 spinal cord was dissected out, rapidly frozen in powdered dry ice, and cut on a cryostat to a thickness of 14 μm. Sections were mounted onto MAS-coated glass slides (Matsunami, Osaka, Japan).

After washing in Tris-buffered saline (TBS) (0.1 M Tris-HCl, pH 7.4, and 0.15 M NaCl), the sections were immersed in 50% ethanol for 10 min, 70% ethanol for 10 min, and 50% ethanol for 10 min to enhance antibody penetration.

For single immunohistochemistry (IHC) staining of c-Fos, the sections were preincubated in TBS containing 5% normal horse serum (NHS) for blocking for 30 min, followed by incubation with polyclonal rabbit anti-c-Fos antibody (1:500, Synaptic Systems, Germany, Cat# 226 003, RRID: AB_2231974) in 5% NHS for 2 h at room temperature. The sections were washed in TBS and then incubated in ImmPRESS horse anti-rabbit IgG Polymer reagent (Vector Laboratories, Cat# MP-7401, RRID: AB_2336529) for 30 min at room temperature. The sections were washed in TBS and the peroxidase reaction was developed with 0.05% diaminobenzidine (DAB) (Wako) and 0.01% hydrogen peroxide in TBS. The sections were then washed in TBS and dehydrated in a graded ethanol series, cleared in xylene, and coverslipped.

For double-labeling IHC of c-Fos with paired box 2 (Pax2), the sections were processed as previously described. ¹⁷ In brief, TBS containing 5% NHS was used for the incubation of sections with guinea pig anti c-Fos polyclonal antibody (1:200, Synaptic Systems, Germany, Cat# 226 308, RRID: AB_2905595) and rabbit anti-Pax2 monoclonal antibody (1:10,000, abcam, UK, Cat# ab79389, RRID: AB_1603338) for 24 h. Fluorescence-conjugated secondary antibodies, donkey anti-rabbit IgG Alexa Fluor 488 (1:1,000, Thermo Fisher Scientific, USA, Cat# A-21206, RRID: AB_2535792) and donkey anti-guinea pig IgG CF555 (1:500, Biotium, USA, Cat# 20276–1, RRID: AB_10853597) were used for the visualization of immunoreactivity. For quantification of c-Fos immunoreactive cells, the immunoreactive intensity was measured by a computerized image analysis system (NIH Image, version 1.61). In 256 Gy scale signal gradients, we considered signal intensities above 192 as positive signals. For the fluorescent image of c-Fos and Pax2, signals on the DAPI were counted as the positive signal.

In situ hybridization histochemistry (ISHH)

Using enzyme-digested clones, ³⁵S UTP-labeled rat GRP and GRPR cRNA probes (GRP: accession number M31176.1, 418–974; in pCRII TOPO vector, GRPR: accession number NM_012706.2, 566–1166; in pCRII TOPO vector) were synthesized. For autoradiography, the sections were coated with NTB emulsion (Eastman Kodak, Rochester, NY, USA), diluted 1:1 with distilled water at 42°C, and exposed for 4 weeks (GRP) or 6 weeks (GRPR) in light-tight boxes at 4°C. After development in D-19 and fixation in 24% sodium thiosulfate, the sections were rinsed in distilled water, stained with hematoxylin, dehydrated in a graded ethanol series, cleared in xylene and coverslipped.

In order to examine the distribution of c-Fos, and GRP or GRPR mRNA, we combined IHC with ISHH. The treatment of sections and the method of double-labeling with IHC and ISHH were described previously. ¹⁸ The sections were washed in TBS and immersed in 50% ethanol for 10 min, 70% ethanol for 10 min, and 50% ethanol for 10 min to enhance antibody penetration, and 0.3% H₂O₂ in methanol for 5 min to block endogenous peroxidase activity. After washing in TBS, the sections were preincubated in 2.5% NHS for 30 min, followed by incubation in rabbit anti-c-Fos antibody (1:500) in 5% NHS with 100 U/mL RNase inhibitor overnight at 4°C. The sections were washed in TBS and then incubated in ImmPRESS horse anti-rabbit IgG Polymer reagent for 30 min at room temperature. The sections were washed in TBS and the horseradish peroxidase reaction was developed in TBS containing 0.05% DAB and 0.01% hydrogen peroxide. Sections were then washed in TBS. After immunohistochemistry, sections were immediately processed for ISHH.

To measure the number of cells positive for GRP or GRPR mRNAs, neuronal profiles that contained nuclei or c-Fos positivity were used for quantification. Using the ISHH sections with sense probe, we determined the background level of grains. In a diameter 10 μm circle of the ISHH section of sense probe, the average number of grain was 0.35 for GRP ISHH and 0.52 for GRPR ISHH. We used a signal-to-noise (S/N) ratio for each cell in each tissue. Cell nuclei or c-Fos-immunoreactivity (ir) with a density of grains ten-fold the background level or higher (approximately more than four or six grains on the nuclei of c-Fos-ir cells labeled for GRP and GRPR, respectively) were considered positively labeled for GRP or GRPR mRNA.

Drug administration

Gastrin-releasing peptide peptide (R&D Systems, MN) and RC-3095 (Sigma-Aldrich, MO) were dissolved in sterile water. Under adequate inhalation anesthesia with sevoflurane (3%–5%, in air), the vertebral arch of the L5 vertebra was resected and a 5 mL soft tube (SILASTIC laboratory tubing, O.D.: 0.64 mm; Dow Corning Corporation, MI, USA) filled with saline solution was inserted. The tube was inserted 10 mm into the subarachnoid space (toward the head). For a single intrathecal injection of GRP, the end of the soft tube was ligated and fixed subcutaneously. The next day, under sevoflurane inhalation anesthesia, the ligated tube was cut and GRP was administered using a Hamilton syringe. Furthermore, 0.03 nmol and 0.3 nmol doses of GRP were administered (diluted in saline at 8.5 μg/mL and 85 μg/mL, respectively).^19,20 For intrathecal administration of RC-3095 for 1 day, a pump (MINI-OSMOTIC PUMP MODEL 2001D; DURECT Corporation, CA) was connected to a soft tube and the pump was held subcutaneously with sutures. The dosages of RC-3095 were 0.3 nmol and 3 nmol per day (diluted in saline at 14.2 ng/mL and 142 ng/mL, respectively), and the solutions were injected using a mini-pump.

Behavioral experiments

After injection of formalin into the plantar of the left paw, the rats were immediately returned to the cage. The number of flinching bouts occurring at 5 min intervals for 60 min after injection was recorded. The GRP-treated group received formalin 3 min after GRP administration and the RC-3095-treated group received formalin 24 h after continuous administration using a mini pump as described above, and pain behavior was evaluated.

For the analysis of the CFA model, heat hypersensitivity was tested using the plantar test (7370, Ugo Basile, Italy). Briefly, a radiant heat source beneath a glass floor was aimed at the plantar surface of the hind paw. Three measurements of latency were taken for each hind paw in each test session. The hind paws were tested alternately with greater than 5 min intervals between consecutive tests. The three measurements of latency per side were averaged. All the behavioral experiments were assessed by researchers blinded to the treatment type.

Statistical analysis

All data are presented as the mean ± standard error of mean (SEM). Statistical analyses were performed using Statview (Statview, SAS Institute Inc., RRID: SCR_017411) and/or JMP Pro 15 (JMP, SAS Institute Inc. RRID:SCR_014242). Parametric statistical tests were used to compare the different experimental groups. The paired Student’s t-test was used to assess statistical significance between two groups (Figure 1(b), (d), (g), (j), and (l), and Figure 3(c) and (d)). When more than three groups were compared, one-way non-repeated measures analysis of variance (ANOVA) was used, followed by a Fisher’s Protected Least Significant Difference (PLSD) post-test (Figures 2, 3(a), 4(a) and (b)). A difference was accepted as significant if the probability was less than 5% (p < 0.05).OPEN IN VIEWER

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Figure 2.

The effects of intrathecal administration of RC-3095, an antagonist for GRPR, on formalin-induced spontaneous paw flinching. Formalin was intradermally injected into the hindpaw followed by the administration of RC-3095 at 5 min after formalin injection (0.3 and 3 nmol). The low dose of RC-3095 (0.3 nmol) partially enhanced the formalin-induced pain behavior from 25 min to 35 min after formalin injection. In all graphs, values are represented as mean ± SEM (n = 8 for saline and 3 nmol RC-3095 group, n = 5 for 0.3 nmol RC-3095 group). # indicates p < 0.05 vs. saline group, one-way ANOVA followed by individual post hoc comparisons (Fisher’s PLSD).

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Figure 3.

(a), The effects of intrathecal administration of GRP on formalin-induced spontaneous paw flinching. Formalin was intradermally injected to the hindpaw followed by GRP administration at 5 min after formalin injection (0.3 and 0.03 nmol). Both doses of GRP administration partially reversed the formalin-induced pain behavior from 5 min to 60 min after formalin injection. In all graphs, values are represented as mean ± SEM (n = 8 for saline group, n = 6 for each of the GRP groups). # indicates p < 0.05 vs. saline group, one-way ANOVA followed by individual post-hoc comparisons (Fisher’s PLSD). (b)-(d), Double labeling analysis of c-Fos-ir with Pax2 in the dorsal of paw formalin injection model treated with intrathecal saline or GRP. (b), Representative images of c-Fos-ir (red) with Pax2-ir (green). Arrows indicate double-labeled cells with c-Fos and Pax2. (c), Quantification of the c-Fos-ir in the dorsal horn of formalin injected rats treated with intrathecal saline or GRP (n = 4, 0.1 mm ² from each rats). Y-axis represents total number of c-Fos-ir per animal (mean ± SEM). # indicates significant compared with the saline treatment group (p < 0.05; t-test). (d), Graph shows the percentage of Pax2-ir profiles in the total c-Fos-ir in the dorsal horn of the formalin injection model treated with intrathecal saline or GRP. # indicates significance compared with the saline treatment group (p < 0.05; t-test).

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Figure 4.

Effect of intrathecal administration of GRP on the thermal sensitivity of CFA model rats. GRP (0.03 nmol) was infused into the intrathecal space at 1 day after CFA injection. (a), Paw withdrawal threshold of the side contralateral to CFA injection. GRP injection did not change the thermal sensitivity of the contralateral side. (b), Intrathecal infusion of GRP significantly inhibited the established thermal hyperalgesia. Note that the withdrawal thresholds for thermal stimuli at 0 and 5 min after GRP infusion were not changed. GRP almost completely reversed thermal allodynia at 60 min after infusion. # indicates p < 0.05, by t-test compared with corresponding vehicle controls (saline) (n = 5).

Effect of intrathecal injection of GRPR antagonist on the formalin induced pain behavior

Induction of c-Fos-ir in GRP- and GRPR positive neurons suggested a possible involvement of GRP in the pain processing in the dorsal horn in the formalin model. In order to elucidate the roles of the endogenous GRP-GRPR system in pain processing, we administered two doses of the GRPR antagonist RC-3095 intrathecally and examined pain behavior using the formalin injection model (Figure 2). As previously reported, the formalin-induced flinching occurred in two phases, which were observed at 0–10 min and at 25–60 min after formalin injection (Figure 2, vehicle treated group). The series of RC-3095 treatments did not affect the first phase of pain behavior (0–10 min after injection). However, in the second phase of the pain behavior from 25 to 35 min after formalin injection, both doses of RC-3095 increased the number of flinches (Figure 2). From 35 to 60 min formalin injection, intrathecal injection of RC-3095 showed a similar pain behavior pattern compared to the control group (Figure 2).

Effects of intrathecal GRP injection on behavior in the formalin model

Enhancement of pain behavior by the intrathecal GRPR antagonist RC-3095 (Figure 2) indicates the anti-nociceptive role of GRP in the acute pain state. In order to examine the anti-nociceptive effect of GRP in the dorsal horn, we examined the impact of exogenous administration GRP on pain behavior in the rat formalin model. To this end we intrathecally administered GRP and subsequently injected formalin into the left hind paw, and examined the change in the number of flinches compared to vehicle control (Figure 3(a)). After formalin injection we could observe a number of spontaneous flinches in the pre-injected saline group. For GRP pre-treatment we tested two doses (0.03 and 0.3 nmol), and both doses of the intrathecal GRP treatment apparently suppressed the formalin-induced flinching behavior not only in the first but also in the second phase (Figure 3(a), GRP treated group). This data suggests that itch related to GRP may have an anti-nociceptive effect on pain behavior after formalin injection into the hind paw.

Using the formalin model treated by intrathecal GRP, we performed double labeling analysis of c-Fos with paired box 2 (Pax2), a marker of inhibitory interneurons, and examined the activation of the inhibitory circuit in the dorsal horn (Figure 3(b)). Paw formalin injection with intrathecal saline treatment induced a substantial amount of c-Fos-ir in the dorsal horn (Figure 3(b) left column). Intrathecal GRP injection significantly reduced formalin-induced c-Fos-ir in the spinal cord (Figure 3(b) upper row and (c)). In the dorsal horn of saline-treated rats, about 18% of the formalin-induced c-Fos-ir was co-labeled with Pax2-ir. Surprisingly, the percentage of the Pax2 positive cells with c-Fos-ir was significantly higher in the GRP treated dorsal horn ipsilateral to the formalin injection (Figure 3(b) merged images and (d)). In the dorsal horn contralateral to the formalin injection, we did not detect c-Fos-ir in the saline (data not shown) and GRP treatment groups (Figure 3(b) right column). Indeed, we injected GRP in the subarachnoid space of the lumbar segment of the formalin-injected rats and did not observe spontaneous itch-related or aversive responses in the lower limb area contralateral to the formalin injection. However, in the cervicofacial area, we observed a limited amount of scratching behavior (20–50 times/1 h) during the observation period (∼1 h) in the GRP treatment group. Therefore, we considered that the GRP activated subsets of spinal inhibitory neurons in the presence of acute pain signal, leading to suppression of pain behavior.

Effect of GRP on inflammation-induced thermal hyperalgesia

So far, we found that the acute pain signal activated a part of the itch-related neuronal circuit, and that the intrathecal GRP injection reduced pain behavior associated with the activation of inhibitory neurons in the dorsal horn in this model. Next, we examined the effect of exogenous GRP on the intraplantar CFA injection-induced inflammatory pain model. CFA injection resulted in increased sensitivity to noxious heat, which is mediated by dorsal horn sensitization. ²¹ On the contralateral side of this model, CFA injection did not change the thermal sensitivity at 1 day after the CFA injection. Gastrin-releasing peptide (0.03 nmol) injection did not affect the contralateral paw withdrawal latency (Figure 4(a)). CFA injection induced thermal hyperalgesia that was characterized by the reduction of the withdrawal threshold to thermal stimuli on the ipsilateral side 1 day after injection (Figure 4(b)). Intrathecal infusion of GRP showed a delayed effect on thermal hyperalgesia. Spinal GRP administration did not affect thermal sensitivity at the early time point but significantly reversed thermal hyperalgesia at 60 min after administration (Figure 4(b) purple bars).