A total of 286 adult male Sprague-Dawley (SD) rats (weight, 180–200 g; age, 8–12 weeks) were obtained from the Experimental Animal Center of Jinling Hospital (Nanjing, China). The rats were maintained at 24±1°C, under a 12 h light-dark cycle, with ad libitum access to food and water. The present study followed the International Association for the Study of Pain guidelines for pain research in animals (18), and all animal studies were approved by the Animal Care and Use Committee of Jinling Hospital.

A total of 16 SD rats were randomly divided into four groups (n=4/group), to determine the levels of PDE4A, B, C and D in the lumbar spinal cord. The rats were sacrificed under anesthesia with sodium pentobarbital (40 mg/kg, i.p.; Suolaibao Biotechnology Co,. Ltd., Beijing, China) and lumbar spinal cords (L4 and L5) were harvested and stored at −80°C. Frozen tissues were homogenized in radioimmunoprecipitation assay lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China) and centrifuged at 4°C for 10 min at 10,000 × g. The supernatants were harvested and protein concentrations were determined using the Bradford method (Leagene Biotechnology Company, Beijing, China). Protein samples (100 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to 0.45-µm polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry milk, and were then incubated with the following rabbit anti-rat polyclonal antibodies: Anti-PDE4A (1:1,000; cat. no. ab14607), anti-PDE4B (1:1,000; cat. no. ab14611), anti-PDE4D (1:500; cat. no. ab14613) (Abcam, Cambridge, MA, USA) and anti-PDE4C (1:300; cat. no. PD4C-301AP: Fabgennix International Inc., Frisco, TX, USA) overnight at 4°C. Subsequently, the membranes were incubated with a horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (Ig)G antibody (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (1:1,000; cat. no. 3683; Cell Signaling Technology, Inc.). The proteins were visualized using chemiluminescence reagents provided within an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK) and were exposed to film. Scanning densitometry (ImageJ2x; Rawak Software, Inc.; National Institutes of Health, Bethesda, MD, USA) was used for the semi-quantitative analysis of the data.

siRNA were designed to target the sequences of rat PDE4A (GenBank accession NM_013101), PDE4B (GenBank accession NM_017031) and PDE4D (GenBank accession NM_017032). The BLOCK-iT™ Alexa Fluor Red Oligo (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as a mismatch control. All of the oligonucleotide sequences were examined against the GenBank database using the Basic Local Alignment Search Tool algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), in order to exclude non-specific matches with any unintended nucleotide sequences. The oligonucleotides, which had been synthesized, individually deprotected, and purified using RNase-free high-performance liquid chromatography, were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The siRNA stocks were aliquoted and stored at −20°C, at a concentration of 200 µM in annealing buffer (19). siRNA sequences used in the present study are presented in Table I.

Under sodium pentobarbital (40 mg/kg, i.p.) anesthesia, an intrathecal catheter (PE-10 tubing, 18 cm) was inserted 1–2 cm cephalad into the rat lumbar subarachnoid space at the L4–L5 intervertebral discs. The catheter implantation procedure was successfully performed in 224 of the 270 remaining rats. The tip of the catheter was placed near the lumbar enlargement of the spinal cord (20). The catheter, with ~20 µl dead space, was subcutaneously tunneled and externalized through the skin of the neck region. After 5 days, 2% lidocaine (10 µl; Jinling Pharmaceutical Co., Ltd., Nanjing, China) was injected intrathecally into the rats with no impaired movement. Rats that exhibited lower limb paralysis within 1 min indicated successful catheterization, and those that exhibited neurological deficits resulting from the surgical procedure were excluded from further experiments.

The L5 spinal nerve ligation model was generated according to methods described by Chung et al (21). Briefly, following anesthetization with sodium pentobarbital (40 mg/kg, i.p.), the rats with intrathecal catheters were placed in the prone position. The left paraspinal muscles were separated from the spinous processes at the L4–S2 level under aseptic conditions. The L5 transverse process was carefully removed, in order to identify the spinal nerves, and the L5 spinal nerve was ligated with 7-0 silk thread. In the sham group, the surgical procedure was identical, except that the left L5 spinal nerve was not ligated. The rats were returned to their cages and observed for any signs of motor deficits. None of the rats exhibited motor dysfunction after surgery.

The rats were divided into seven groups (n=32/group): The sham group (sham surgery + saline), the saline group (SNL + saline), the vehicle group (SNL + Lipofectamine^® RNAiMAX; Invitrogen; Thermo Fisher Scientific, Inc.), the mismatch siRNA group (SNL + mismatch siRNA), the PDE4A-siRNA group (SNL + PDE4A-siRNA), the PDE4B-siRNA group (SNL + PDE4B-siRNA) and the PDE4D-siRNA group (SNL + PDE4D-siRNA). siRNA or mismatch RNA complexes were prepared immediately prior to administration by mixing the RNA solution with Lipofectamine^® RNAiMAX transfection reagent, at a ratio of 1:4 (w:v). The final concentration of siRNA was 2 µg in 10 µl. siRNAs, mismatch RNA, saline or Lipofectamine^® RNAiMAX were administered intrathecally just after ligation, and at 1, 3, 5 and 7 days after surgery.

Thermal and mechanical hyperalgesia were measured 1 day prior to and 2, 4, 6 and 8 days after surgery. Behavioral studies were carried out in a quiet, temperature-controlled (24°C) room between the hours of 8:00 AM and 10:00 AM.

Mechanical withdrawal threshold (MWT) was measured using an Electro Von Frey anesthesiometer (Model 2390CE; IITC, Inc., Woodland Hills, CA, USA). Briefly, the rats were individually placed beneath an inverted ventilated Plexiglas cage with a metal-mesh floor, allowing access to the plantar surface of the hind paw. After 30 min of acclimation, gentle incremental pressure (maximum 200 g) was applied using a rigid von Frey hair to the plantar surface of the ipsilateral hind paw, until the paw was withdrawn. Five tests were conducted at intervals of 5 min and the force (g) applied was recorded.

Thermal withdrawal latency (TWL) was determined using radiant heat (Model 390; IITC, Inc.). Following acclimation to the Plexiglas cage (23×18×13 cm; 3-mm-thick glass floor), the radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw when in contact with the floor. The paw TWL was obtained five times per animal with intervals of 5 min. Light intensity was preset, in order to obtain a baseline latency of ~10 sec and the cutoff time was set at 20 sec to avoid tissue damage.

Following behavioral testing at 2, 4, 6 and 8 days after the operation, all of the rats were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and were sacrificed by decapitation, and all appliances were treated with diethylpyrocarbonate to prevent the degradation of mRNA. The lumbar spinal cords were harvested and the mRNA expression levels of PDE4A, B and D were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (n=6 at each time point for each group). The protein expression levels of PDE4A, B and D were assessed by western blotting, as previously described.

Following homogenization of the tissue samples, total RNA was extracted from the ipsilateral lumbar spinal cord using an RNA Isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA was detected using a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). cDNA was synthesized by reverse transcription (RT). The RT reaction was carried out in a 20 µl total reaction volume, containing 4 µl 5X RT buffer, 4 µl 2.5 mM dNTPs, 1 µl Multiscribe reverse transcriptase (50 U/µl) (Promega Corporation, Madison, WI, USA), 1 µl RNase inhibitor, 5 µl RNase-free water, and 3 µg DNase-treated total RNA in a 5 µl volume. The RT reaction was carried out at 25°C for 10 min, 37°C for 120 min, and 95°C for 5 min. qPCR was conducted using a Rotor-Gene 3000 Real Time PCR system (Qiagen, Inc., Valencia, CA, USA). The sequences of the primers (Invitrogen; Thermo Fisher Scientific, Inc.) used in the present study are presented in Table II. qPCR was performed using SYBR Green I (1:20,000; Qiagen, Inc., Valencia, CA, USA), with the following cycling conditions: 1 cycle at 95°C for 3 min, followed by 40 cycles at 95°C for 45 sec, 61°C for 45 sec, 72°C for 40 sec and 80°C for 5 sec. The PCR reaction volume (25 µl) consisted of 3 units Platinum Taq DNA polymerase, 1.5 mM MgCl₂, 205 µM dGTP, dCTP, dATP and dTTP, 400 nM forward and reverse primers, 2.5 µl 10X PCR buffer (all Promega Corporation), 10 µl SYBR Green I and 1 µl cDNA. The mRNA expression levels were calculated according to relative standard curves. The curves were generated by plotting the quantification cycle (Cq) against the log amount of total cDNA added to the reaction. The relative target gene expression levels were determined using the 2^−ΔΔCq method (22). Results were normalized to GAPDH.

A total of 8 days after surgery, the protein expression levels of ERK, p-ERK, CD11 and GFAP were detected by western blotting (n=4), as described previously in the present study. For ERK and p-ERK, 50 µg samples were separated in each lane. The following antibodies were used: Anti-ERK1/2 (1:1,000; cat. no. 9102), anti-p-ERK1/2 (Thr202/Tyr204) (1:1,000; cat. no. 9101) (Cell Signaling Technology, Inc.), anti-GFAP (1:500; cat. no. sc-9065; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-CD11b (1:1,000; cat. no. ab75476) and HRP-conjugated goat anti-mouse (cat. no. ab47827)/anti-rabbit (cat. no. ab6721) Ig (1:1,000) (Abcam).

The expression of cytokines, including TNF-α, IL-1β, IL-6 and IL-10, was determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) 8 days after surgery (n=4/group), according to the manufacturer's protocol. Ipsilateral lumbar spinal cord samples (40–50 mg) were dissected and homogenized in a buffer containing a protease inhibitor (Roche Diagnostics, Mannheim, Germany) using a Power Gen 124 tissue tearer (Thermo Fisher Scientific, Inc.). The samples were then centrifuged at 20,000 × g for 30 min at 4°C. The supernatants were aliquoted and stored at −80°C for further protein quantification.

Data are presented as the mean ± standard deviation. Data from the western blotting and RT-qPCR studies were analyzed using one-way analysis of variance (ANOVA). Data from the thermal and mechanical hyperalgesia tests were analyzed using two-way ANOVA, in order to determine the differences between the groups at various time points. All statistical analyses were performed using SPSS 18.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

PDE4A, B and D protein expression was detected in the spinal cord of naïve rats (Fig. 1); however, PDE4C expression was not detected (data not shown).

PDE4A, B and D mRNA expression levels were significantly upregulated 2, 4, 6 and 8 days after L5 SNL, as compared with the sham group (Fig. 2). Treatment with saline, vehicle or mismatched RNA did not reduce the upregulated mRNA expression levels (P<0.05; Fig. 2). However, treatment with 2 µg siRNA-PDE4A, B or D significantly decreased the mRNA expression levels ofPDE4A, B or D, respectively (P<0.05; Fig. 2), as compared with the saline group. This effect, first observed at 2 days post-L5 SNL, continued to 8 days post-ligation without significant alterations.

siRNA exhibited similar effects on the protein expression levels of PDE4A, B and D (Fig. 3). L5 ligation resulted in overexpression of PDE proteins, and intrathecal injection with siRNA-PDE4A, B or D significantly lowered the expression of PDE4A, B or D, respectively.

Prior to L5 SNL, all groups exhibited comparable baseline thresholds for mechanical and thermal stimuli (Fig. 4A and B; -1 day). The values of the sham group rats were steady during the experimental period (Fig. 4A and B). L5 SNL led to significantly reduced MWT and TWL (P<0.05; Fig. 4). No significant differences were observed between the saline, vehicle and mismatched RNA groups (P>0.05; Fig. 4). In addition, intrathecal injection with PDE4A-siRNA and PDE4D-siRNA did not improve the mechanical allodynia and thermal hyperalgesia, as compared with the saline group (P>0.05; Fig. 4). However, injection with PDE4B-siRNA significantly attenuated the development of mechanical allodynia and thermal hyperalgesia, as compared with the saline group (P<0.05; Fig. 4).

L5 SNL resulted in a significant increase in the expression levels of p-ERK, as compared with the sham group (Fig. 5A; P<0.05). Compared with the saline group, only the PDE4B-siRNA group exhibited a significant decrease in the expression levels of p-ERK (Fig. 5A; P<0.05). However, there was no difference in the expression of total ERK between the various groups (Fig. 5B; P>0.05).

CD11b, a microglial marker (Fig. 6A) and GFAP, an astrocyte marker (Fig. 6B), were measured 8 days after L5 SNL. CD11b and GFAP protein expression levels were higher following SNL, as compared with the sham group (P<0.05; Fig. 6). However, intrathecal injection of PDE4B-siRNA attenuated the upregulated expression of CD11b and GFAP, as compared with the saline group (P<0.05; Fig. 6).

To investigate the effects of PDE4B-siRNA, the expression levels of numerous inflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-10, were measured. Compared with the sham group, the expression levels of TNF-α, IL-6 and IL-1β were significantly increased in the SNL groups (P<0.05). However, the PDE4B-siRNA group exhibited significantly reduced levels of the proinflammatory cytokines, as compared with the saline group (P<0.05). In addition, PDE4B-siRNA significantly enhanced IL-10 production, as compared with the other SNL groups (P<0.05; Table III).