Male SPF Sprague-Dawley rats (200–250 g) were obtained from the Experimental Animal Center of Guangdong Medical University (Guangdong, China). All animals were maintained on a 12 h light/dark cycle at a room temperature of 22±1°C with food and water available ad libitum. All experimental protocols described in the present study were approved by the Animal Care and Use Committee at Guangdong Medical University and conformed to the NIH guidelines (10).

Peripheral neuropathic pain was induced in the rats via the administration of 50 µg/kg vincristine sulfate intraperitoneally (i.p.) for 10 consecutive days, as described by Siau and Bennett (11).

Animals were anesthetized with 60 mg/kg, intraperitoneal injection (i.p.) of sodium pentobarbital. A polyethylene (PE)-10 tube (BD Biosciences, San Jose, CA, USA) was implanted into the subarachnoid space of the lumbar enlargement for intrathecal drug administration. The catheter placement was verified by observing transient hindpaw paralysis induced by intrathecal injection of 2% lidocaine (10 µl). Only those rats showing complete paralysis of both hind limbs and the tail following the administration of lidocaine were used for the subsequent experiments. At the end of each experiment, the position of the PE tubing in the intrathecal space at the lumbar enlargement was visually verified by exposing the lumbar spinal cord.

Vincristine sulfate (Hisun Pharmaceutical Co., Ltd., Zhejiang, China) was dissolved in normal saline (0.9% NaCl). Fucoidan from Fucus vesiculosus (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was dissolved in normal saline (0.9% NaCl). Different doses of fucoidan (50, 100 and 200 mg/kg) were injected i.p. The day prior to the first administration of vincristine was considered to be day 0. The investigation was divided into three experiments. In the single treatment experiment, fucoidan (50, 100 or 200 mg/kg) or normal saline was administered to the vincristine-treated rats once on day 14 (n=6 rats per treatment group). Behavioral assessments were performed on day 14 immediately prior to administration of the drug, and at 2, 12 h, 1 day, 3 days and 7 days post-administration.

In the repetitive treatment experiment, the animals were divided into six groups for administration: Rats were administered with normal saline for 14 consecutive days as a control group; vincristine-treated rats were administered with fucoidan (50, 100 or 200 mg/kg i.p.) or normal saline once daily for 14 consecutive days; pregabalin (Sigma-Aldrich; Merck Millipore) was dissolved in normal saline and 10 mg/kg was injected (i.p.) for 14 consecutive days in the vincristine-treated rats as a positive control (n=6 rats per treatment group). Behavioral assessments were performed on days 0, 1, 3, 7, 14 and 21.

To evaluate the involvement of GABAB receptor in the analgesic effect of fucoidan, a third experiment was performed. Based on the results obtained in the repetitive treatment experiment, a 200 mg/kg/day dose of fucoidan was selected for further investigations. The vincristine-treated rats were administered with 200 mg/kg fucoidan or normal saline once daily for 14 consecutive days. Saclofen (Sigma-Aldrich; Merck Millipore) was diluted to a concentration of 1 µg/µl in saline for intrathecal injection. Between days 15 and 21, 10 µl of saclofen or saline were administered once daily for 7 consecutive days. A total of 24 rats were equally randomized into four groups: i) Vincristine-saline-saline treatment; ii) vincristine-fucoidan-saline treatment; iii) vincristine-saline-saclofen treatment; (4) vincristine-fucoidan-saclofen treatment. Behavioral assessments were performed on days 14 and 21.

Mechanical allodynia was assessed using von Frey filaments (Stoelting, Kiel, WI, USA) by experimenters who were blinded to the group assignment. The ipsilateral hind paw was pressed with one of a series of von Frey filaments with gradually increasing rigidity (2, 4, 6, 8, 10, 15 and 20 g), each of which was applied to the plantar surface for 5–6 sec. A positive paw withdrawal response was recorded if the animal briskly lifted the hindpaw. The mechanical withdrawal threshold was determined using the up-down method (12).

Cold allodynia was assessed by placing 100 µl of acetone on the planter surface of the left hind paw of the rat. A cold chemical sensitive reaction with respect to either paw licking, shaking or rubbing the left hind paw was observed and recorded as the paw withdrawal threshold. The cut-off duration of 20 sec was maintained.

Following the behavioral assessments on day 21, the rats were anesthetized with chloral hydrate (300 mg/kg, i.p.), perfused intracardially with 250 ml cold saline and then sacrificed by decapitation. The L4-6 spinal cord was promptly removed onto an ice-cold plate, frozen and stored at −80°C until use. All collected tissue samples were homogenized in an SDS sample buffer containing a mixture of proteinase and phosphatase inhibitors (Sigma-Aldrich; Merck Millipore). Each individual rat spinal cord protein was processed separately. The protein concentrations were estimated using the bicinchoninic acid method. Protein samples (30 µg) were separated by 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were blocked in 10% skimmed milk for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4°C: Guinea pig polyclonal anti-GABAB2 (1:2,000; cat. no. AB2255; Chemicon, Temecula, CA, USA) and mouse monoclonal anti-β-actin (1:3,000; cat. no. A1978; Sigma-Aldrich; Merck Millipore). The blots were further incubated for 1 h at room temperature with HRP-conjugated secondary antibody (anti-guinea pig 1:3,000; cat. no. 61_4620; anti-mouse cat. no. 31430; 1:5,000; GE Healthcare Life Sciences, Chalfont, UK), and washed three times in TBS for 30 min. The immune complexes were detected using the enhanced chemiluminescence detection method (GE Healthcare Life Sciences) and exposure to film. A square of the same size was drawn around each band to measure the density, and the background adjacent to that band was subtracted. Levels of the target protein were normalized against the levels of β-actin and expressed as relative fold changes.

Values are expressed as the mean ± standard error of the mean. For behavioral data, comparisons were made using two-way repeated measures analysis of variance (ANOVA). For others, comparisons were performed using two-way ANOVA followed by Bonferroni tests. Data were analyzed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Vincristine administration resulted in the development of mechanical and cold allodynia, as reflected by the significant reductions in the mechanical and cold withdrawal thresholds, compared with the normal control group. To determine the effects of a single injection of fucoidan on vincristine-induced allodynia, fucoidan was injected i.p. on day 14, when the neuropathic pain had been maintained. The behavioral responses 2, 12 h, 1 day, 3 days and 7 days following administration were then observed. No differences in mechanical or cold allodynia were detected between the rat treated with fucoidan and normal saline, at any doses or time points, following injection (Fig. 1A and B).

The effects of repeated fucoidan treatment on vincristine-induced neuropathic pain were also examined. As shown in Fig. 2A and B, repeated administration of fucoidan attenuated vincristine-induced mechanical and cold allodynia in a dose-dependent manner. A lower dose of fucoidan (50 mg/kg) only marginally elevated the mechanical and cold paw withdrawal threshold. Higher doses of fucoidan (100 and 200 mg/kg) markedly and significantly increased the mechanical and cold paw withdrawal threshold. Similar effects were observed with pregabalin administration. In addition, a higher dose of fucoidan ameliorated vincristine-induced neuropathic pain for at least 1 week following the final treatment.

GABAB receptor is densely expressed in the spinal cord dorsal horn. Activation of spinal GABAB receptor produces antinociception in acute and chronic pain (13,14). A study by Thibault et al (15) reported that the GABAB receptor is functionally required for the alleviating effect of oxycodone in vincristine-induced neuropathic pain. The analgesic effect of fucoidan on vincristine-induced neuropathic pain may be associated with its upregulation of the expression of GABAB receptor. To confirm this hypothesis, the present study examined the expression levels of GABAB2 receptor using western blot analysis. The data showed that there was an upregulation in the expression of GABAB2 receptor in the vincristine-fucoidan treated animals, compared with the expression in the animals in the vincristine-saline treated animals or the saline-treated animals on 21 (Fig. 3A and B).

The present study directly examined the role of GABAB receptor in the analgesic effect of fucoidan by assessing the effect of the intrathecal administration of saclofen, a selective GABAB receptor antagonist. As shown in Fig. 4A and B, the alleviating effect of repeated fucoidan administration on vincristine-induced mechanical and cold allodynia was completely reversed by the antagonist. Furthermore, saclofen eliminated the effect of fucoidan on the upregulated expression of GABAB2 receptor in the spinal cord (Fig. 5). Of note, the antinoticeptive effect of fucoidan (200 mg/kg) was maintained 7 days following the final administration. These results revealed that fucoidan had a prolonged effect via the upregulation of GABAB receptor, which can be observed even when fucoidan treatment is terminated.