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Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain
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Chuanyin Hu a , Guoping Zhang a , Yun-tao Zhao b,∗ a b
Department of Biology, Guangdong Medical College, Zhanjiang, Guangdong 524023, People’s Republic of China Modern Biochemistry Center, Guangdong Ocean University, Zhanjiang, Guangdong 524088, People’s Republic of China
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h i g h l i g h t s • Fucoidan attenuates the existing allodynia and hyperalgesia induced by SNL. • Fucoidan significantly inhibits cytokines production, glial and ERK activation. • The analgesic effect of fucoidan may be through inhibiting neuroimmune activation.
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Article history: Received 5 February 2014 Received in revised form 15 April 2014 Accepted 24 April 2014 Available online xxx
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Keywords: Fucoidan Neuropathic pain Neuroimmune activation
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1. Introduction
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Fucoidan is an active constituent found in brown seaweeds, which have potential neuroprotection. The current study aimed to investigate the effects of fucoidan on the maintenance of neuropathic pain induced by L5 spinal nerve ligation (SNL) and the underlying mechanism related to the spinal neuroimmune responses. Animals were randomized into 5 groups: sham-operation with vehicle and SNL with vehicle or fucoidan (15, 50, and 100 mg/kg). Different doses of fucoidan or vehicle were administered intrathecally once daily from postoperative day (POD) 11–20. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) was measured on 1 day before operation and days 10, 20, 22, 24, 26, 28, 30 after operation. Glial activation markers such as glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1 (mac-1), inflammatory cytokines such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1 and IL-6 activation, and extracellular signalregulated protein kinase (ERK) activation in the lumbar spinal cord were determined on day 30 after operation. The results showed that fucoidan caused dosedependently attenuation of mechanical allodynia and thermal hyperalgesia. Furthermore, fucoidan could markedly inhibit neuroimmune activation characterized by glial activation, production of cytokines as well as ERK activation. The analgesic effect of intrathecal fucoidan in rats receiving SNL might partly attribute to the inhibition of neuroimmune activation associated with the maintenance of neuropathic pain. © 2014 Published by Elsevier Ireland Ltd.
Neuropathic pain develops as a consequence of a lesion or disease affecting the somatosensory pathways in the peripheral or central nervous system, and occurs in many neurological diseases. Accumulating evidence indicates that central neuroimmune activation and neuroinflammation play a pivotal role in the generation and maintenance of neuropathic pain [6,15]. After nerve injury, spinal microglia and astrocytes are activated and release pro-inflammatory cytokines, including interleukin (IL)-1, IL-6,
∗ Corresponding author. Tel.: +86 759 2396039; fax: +86 759 2396039. E-mail address: [email protected] (Y.-t. Zhao).
tumor necrosis factor-alpha (TNF-␣) and chemokines such as CCchemokine ligand (CCL) 2, CCL3, CCL5, CCL12 and fractalkine. These cytokines and chemokines elicit chronic neuroinflammation and enhance the central immune cascade, leading to neuropathic pain [10,19]. Furthermore, activated glia cells interact with neurons associated with pain transmission, and increase the excitability of these neurons, resulting in neuropathic pain [10,14]. Neuropathic pain is characterized by spontaneous pain, hyperalgesia and allodynias, affecting millions of people worldwide [24]. It is often severely debilitating and largely resistant to treatment. Currently, available therapies shown to be effective in managing neuropathic pain include opioids and tramadol, anticonvulsants, antidepressants, topical treatments (lidocaine patch, capsaicin), and ketamine. However, these treatments are either relatively
http://dx.doi.org/10.1016/j.neulet.2014.04.030 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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ineffective or accompanied by substantial side effects [16,20]. Therefore, it is urgent to clarify the molecular mechanisms underlying neuropathic pain and establish novel therapeutic strategies. Fucoidan is a sulfated polysaccharide which is one of the main constituents of brown seaweeds. Fucoidan exhibits a variety of biological activities, including antioxidant, anti-apoptosis, anticoagulant, antitumor, antiviral and anti-inflammatory activity [1,21]. However, it was still unclear whether fucoidan had beneficial effects in treating neuropathic pain. In the present study, we tested whether intrathecal fucoidan could attenuate nocifensive behavior associated with neuropathic pain. We tried to explore the underlying mechanism of the potential analgesic effect of fucoidan.
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2. Materials and methods
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2.1. Animals
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Male SPF Sprague-Dawley rats (200–250 g) were used in the present study. All animals were housed under standardized conditions in a room on a 12 h light/dark cycle with food and water available ad libitum. All experimental protocols described in this study were approved by the regulations stipulated by Guangdong Medical College Animal Care Committee which follows the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1985).
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2.2. Surgical operation
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Peripheral nerve injury was produced by SNL according to a previously described method [11]. Briefly, under chloral hydrate anesthesia (300 mg/kg, intraperitoneally, Sigma–Aldrich), a skin incision (3–4 cm) was made in the midline lumbar region (L4–S1). The L6 transverse process was identified, freed of muscular attachments, and partially removed with the help of bone ronguers. This exposed the L5 spinal nerve. The L5 was then carefully isolated and tightly ligated distal to the dorsal root ganglion (DRG) with 6–0 silk thread. The surgical procedures for the sham group were identical to that of the SNL group, except that the spinal nerves were not ligated.
2.3. Intrathecal implantation Polyethylene (PE) – 10 tube (BD, 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 L). Only those rats showing complete paralysis of both hind limbs and the tail after 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.
2.5. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) Behavioral tests were carried out between 8 and 12 am on 1 day before operation and days 10, 20, 22, 24, 26, 28, 30 after operation, respectively. Mechanical allodynia was assessed using von Frey filaments (Stoelting, Kiel, WI, USA) by experimenters blind to group assignment. The stiffness of the von Frey filaments was 2, 4, 6, 8, 10, 15, and 20 g. A positive paw withdrawal response was recorded if the animal briskly lifted the hindpaw. The mechanical withdrawal threshold (MWT) was determined by the up-down method [2]. To assess thermal sensitivity, paw-withdrawal latencies to a radiant heat stimulus were measured using the paw-flick test apparatus (IITC Life Science, Woodland Hills, CA). The thermal withdrawal latency (TWL) was recorded as the threshold of thermal sensitivity. Each hindpaw was tested five times at 5 min intervals. 2.6. Tissue harvest After finishing the behavioral tests on day 30 post operation, rats were deeply anesthetized with chloral hydrate (300 mg/kg, intraperitoneally) and then perfused intracardially with 250 ml cold saline. The ipsilateral L4–5 spinal cord tissue rostral to the injury site was removed, dissected on cold ice and removed quickly to liquid nitrogen for subsequent assays. 2.7. Real-time quantitative polymerase chain reactions Glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1 (mac-1) were markers for astrocytic and microglial activation respectively. Rats were deeply anesthetised with chloral hydrate (300 mg/kg) and sacrificed. Total RNA from the L5 spinal segments was extracted with Trizol (GIBCO/BRL Life Technologies Inc., Grand Island, NY, USA). Complementary DNA (cDNA) was synthesized with oligo (dT) 12–18 using Superscript III Reverse Transcriptase for RT-PCR (Invitrogen, Carlsbad, CA, USA). The sense primer for GFAP was 5 -GCTGGAGCAAGACAAACATTC-3 , and the antisense primer was 5 -CCCTACCCACTCCTACATCGT-3 . The sense primer for mac-1 was 5 -TGGCTGCTACTACCGTCCCT-3 , and the antisense primer was 5 -CAGGTTGGAGCCGAATAGGT-3 . The sense primer for GAPDH was 5 -CCCCCAATGTATCCGTTGTG-3 , and the antisense primer was 5 -TAGCCCAGGATGCCCTTTAGT-3 . Equal amounts of RNA (1 g) were used to prepare cDNA using the SYBR Premix Ex Taq TM (Takara, Tokyo, Japan) and analyzed with a realtime PCR detection system (Applied Biosystems, Foster City, CA, USA). All values were normalized to GAPDH expression. 2.8. Enzyme-linked immunosorbent assay Spinal production of TNF-␣, IL-1 and IL-6 was quantified by enzyme-linked immunosorbent assay (ELISA) kits for rats according to the manufacturers’ instructions (Biosource USA for IL-1 and IL-6; R&D Systems for TNF-␣). Values were expressed as picogram per milligram protein. 2.9. Western blot analysis
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2.4. Drug administration Fucoidan (Sigma, St. Louis, MO) was dissolved in 5% dimethylsulfoxide (DMSO) and then diluted with 0.9% (w/v) saline solution. Animals were divided into 5 groups for administration: shamoperation with vehicle (sham group); SNL rats received treatment with fucoidan (15 mg/kg, 50 mg/kg, 100 mg/kg) or vehicle (5% DMSO) once daily during the period of days 11–20 inclusively (n = 6 rats per treatment group).
To examine changes in p-ERK expression following SNL, all collected tissue samples were homogenized in an SDS sample buffer containing a mixture of proteinase and phosphatase inhibitors (Sigma). The protein concentrations were estimated using the bicinchoninic acid (BCA) method. Protein samples were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked in 10% skimmed milk for 1 h at room temperature and incubated with the following primary antibodies overnight at 4 ◦ C: rabbit anti-pERK (1:1000; Cell Signaling)
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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and mouse anti--actin (1:3000, Sigma). The blots were further incubated for 1 h at RT with HRP-conjugated secondary antibody (anti-rabbit 1:3000, anti-mouse 1:5000; Amersham Biosciences), and washed three times in TBS for 30 min. The immune complexes were detected by the enhanced chemiluminescence (ECL) detection method (Amersham) and exposure to film. A square of the same size was drawn around each band to measure the density and the background near that band was subtracted. Target protein levels were normalized against -actin levels and expressed as relative fold changes compared to the sham group.
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2.10. Statistical analysis
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Values are expressed as means ± SEM. For behavioral data, comparisons were performed using repeated measures of analysis of variance (ANOVA). For others, comparisons were performed using ANOVA followed by Bonferroni tests. Data were analyzed using SPSS 13.0 (Chicago, IL, USA). P < 0.05 was considered significant.
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3. Results
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3.1. Intrathecal administration of fucoidan attenuates SNL induced mechanical allodynia and thermal hypersensitivity in a dose-dependent manner
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To investigate whether fucoidan would reverse established neuropathic pain, different doses (15, 50 and 100 mg/kg) of fucoidan were injected intrathecally once daily from postoperative day (POD) 11–20, and behavioral experiments were carried out. As shown in Fig. 1, the sham group did not display significant behavioral changes. Prominent mechanical and thermal hyperalgesic behaviors were established on day 10 after surgery as shown in the SNL-Veh group. Compared to the SNL-Veh group, treatment of fucoidan dose-dependently alleviated and reversed mechanical allodynia and thermal hyperalgesia. Especially, significant analgesic effect was observed when fucoidan was at doses of 50 and 100 mg/kg.
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3.2. The effects of fucoidan on SNL-induced spinal glial activation
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The analgesic effect of fucoidan might be associated with its inhibitory effect on microglia and astrocytes. To verify this hypothesis, we studied the expression of glial activation markers in lumbar spinal cord GFAP (Fig. 2A) and mac-1 (Fig. 2B) expression. Results showed that on day 30 after SNL, GFAP and mac-1 expression levels were markedly enhanced in SNL rats compared to the shamoperated rats (P < 0.01). Administration of various doses of fucoidan decreased spinal glial activation markers in a dose-dependent manner compared to the SNL-Veh group. Fucoidan, especially in the dose of 50 and 100 mg/kg, seems to exert higher suppressive effects. Furthermore, fucoidan suppression of spinal glial activation was accompanied by its attenuation of behavioral hyperalgesia.
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3.3. The effects of fucoidan on inflammatory cytokines
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The inhibitory effect of fucoidan on glia could lead to subsequent decreased production of inflammatory cytokines. Inflammatory cytokine (TNF-␣, IL-1, and IL-6) expression was analyzed by ELISA (Fig. 3). Results indicated that SNL surgery induced remarkable increases of inflammatory mediators including TNF-␣, IL-1 and IL-6 in the spinal dorsal horn on day 30 after surgery. However, intrathecal fucoidan significantly suppressed the elevations of these inflammatory mediators in a dose-dependent manner. Additionally, there was a significant downregulation of proinflammatory cytokines in the dose of 50 and 100 mg/kg fucoidan treatment group. Moreover, fucoidan inhibition of inflammatory
Fig. 1. The effects of intrathecal fucoidan on SNL-induced mechanical allodynia and thermal hyperalgesia. SNL injury resulted in prominent mechanical allodynia (A) and thermal hyperalgesia (B) on day 10 after surgery. The intrathecal administration of fucoidan (15, 50 or 100 mg/kg, once per day from POD 11–20) significantly blocked SNL-induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. SNL: spinal nerve ligation; POD: postoperative day. n = 6 in each group, *P < 0.05, compared to the SNL + Veh group.
cytokines expression was in line with its alleviation of hypersensitivity.
3.4. SNL-induced phosphorylation of ERK in the spinal dorsal horn is significantly inhibited by fucoidan Substantial evidence implicated that activation of mitogenactivated protein kinase (MAPK) initiates many signaling cascades and increases the synthesis of proinflammatory mediators following peripheral and central nervous injuries [7,17]. To examine whether MAPK activation was involved in the suppressive effect of fucoidan on SNL-induced inflammatory cytokines elevation, we assessed the levels of phosphorylation of ERK using western blot analysis. Our data showed that on day 30 after SNL, the phosphorylation level of ERK was significantly increased in SNL-vehicle group compared to sham-vehicle group (Fig. 4). Similar to the effect of fucoidan on inflammatory cytokines expression, the SNL-induced elevation of the phosphorylation level of ERK was also attenuated by intrathecal fucoidan.
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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Fig. 2. Expression changes of glial activation markers, GFAP and mac-1 in the lumbar spinal cord of rats on day 30 after the operation. SNL increased GFAP (A) and mac-1 (B), while fucoidan dose-dependently down-regulated the markers mentioned above. n = 6 in each group, *, **: P < 0.05, P < 0.01 compared to the sham + Veh group. #, ##: P < 0.05, P < 0.01 compared to the SNL + Veh group.
Fig. 3. The effects of intrathecal fucoidan on the proinflammatory associated cytokines. The protein levels of the proinflammatory cytokines TNF-␣ (A), IL-1 (B), and IL-6 (C) were determined in the spinal dorsal horn in rats that received different treatments using ELISA. Fucoidan significantly prevents and reverses SNL-induced increases of these inflammatory mediators. n = 6 in each group, *, **: P < 0.05, P < 0.01 compared to the sham + Veh group. #, ##: P < 0.05, P < 0.01 compared to the SNL + vehicle group. The levels of proinflammatory cytokines are expressed as relative fold changes compared to the Sham + Veh group.
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4. Discussion In the present study, we observed that repeated administration of fucoidan dose-dependently attenuates the existing mechanical allodynia and thermal hyperalgesia induced by SNL. Subsequently, we investigated the mechanisms that underlie the analgesic effect of fucoidan. We found that intrathecal fucoidan could block the maintenance of neuropathic pain. Furthermore, fucoidan significantly inhibits spinal astrocytic and microglial activation, proinflammatory mediators production and MAPK activation. Our results suggest that
fucoidan may have therapeutic potential in attenuating neuropathic pain, possibly through inhibiting spinal neuroimmune activation. Activation of MAPK has been implicated in a number of signaling events that are important for the initiation and maintenance of neuropathic pain. In the SNL neuropathic pain model, it has been reported that MAPK including ERK, p-38 and JNK are activated in microglia and or astrocyte [8,10,22,23]. We also observed ERK activation in the current study. After repeated intrathecal fucoidan, the phosphorylation level of ERK declined. A previous study has shown that partial sciatic nerve ligation (PSNL) increased p38-MAPK
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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Fig. 4. The effects of intrathecal fucoidan on SNL-Induced ERK activation. The expression of phosphorylated (p) ERK (A and B) was revealed by Western blot analysis. On day 30 after SNL, the phosphorylation level of ERK is significantly increased in SNL vehicle rats. After treatment with fucoidan (100 mg/kg) from SNL day 11 to day 20, the phosphorylation level is remarkably decreased. n = 6 in each group, *P < 0.05 compared to the sham group. #P < 0.05 compared to the SNL + vehicle group. The results are expressed as relative fold changes compared to the Sham + Veh group.
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activation [13]. However, clonidine suppressed activation of p38 MARK in sensory neurons after nerve injury [4]. As inhibition of the MAPK signaling pathway could contribute to inflammation response, the suppression of MAPK activation by fucoidan further added to its role on neuropathic pain and the associated neuroimmune activation. Neuroimmune activation including glial activation and inflammatory cytokine release contributes to neuropathic pain. After peripheral and central nervous injuries, astrocytes and microglia are activated. Then these glial cells release inflammatory cytokines, which amplify neuronal excitation and pain signaling. Therefore, drugs that suppress glial activation and inflammatory cytokines production may prevent and reverse neuropathic pain. In this study, we observed that fucoidan suppressed spinal glial activation and proinflammatory cytokines production. The inhibitory effects of fucoidan on neuroinflammation are in agreement with several other studies on the effects of fucoidan on inflammation diseases. It has been shown that fucoidan significantly inhibited lipopolysaccharide (LPS)-activated microglial inflammatory responses [18]. Moreover, it inhibited the expression of some cytokine/chemokine in LPS accelerated cerebral ischemic injury rats [9]. In addition, fucoidan could ameliorate the learning and memory abilities in A-induced Alzheimer’s disease rats [5]. Fucoidan could also attenuate ischemia-reperfusion (I/R)-induced myocardial damage and prevent the loss of dopaminergic neurons in a LPS-induced animal model of Parkinson’s disease [3,12]. These data suggest that fucoidan has anti-inflammatory activities and neuroprotective effects. It could be considered for use as a potential treatment for inflammatory and neurodegenerative disorders. Acknowledgments
This work was supported by grants from the Doctoral ProQ2 gram of Guangdong Medical College, China (XB1021), the Science 286 and Technology Project Foundation of Zhanjiang City, China 287 (2010C3104009), the Medical Scientific Research Foundation of 288 Guangdong Province, China (B2010234) and the Breeding Project 289 of the Education Department of Guangdong Province, China 290 (LYM10084). We declare no conflicts of interests. 291 285
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Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain
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Chuanyin Hu a , Guoping Zhang a , Yun-tao Zhao b,∗ a b
Department of Biology, Guangdong Medical College, Zhanjiang, Guangdong 524023, People’s Republic of China Modern Biochemistry Center, Guangdong Ocean University, Zhanjiang, Guangdong 524088, People’s Republic of China
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h i g h l i g h t s • Fucoidan attenuates the existing allodynia and hyperalgesia induced by SNL. • Fucoidan significantly inhibits cytokines production, glial and ERK activation. • The analgesic effect of fucoidan may be through inhibiting neuroimmune activation.
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Article history: Received 5 February 2014 Received in revised form 15 April 2014 Accepted 24 April 2014 Available online xxx
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Keywords: Fucoidan Neuropathic pain Neuroimmune activation
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1. Introduction
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Fucoidan is an active constituent found in brown seaweeds, which have potential neuroprotection. The current study aimed to investigate the effects of fucoidan on the maintenance of neuropathic pain induced by L5 spinal nerve ligation (SNL) and the underlying mechanism related to the spinal neuroimmune responses. Animals were randomized into 5 groups: sham-operation with vehicle and SNL with vehicle or fucoidan (15, 50, and 100 mg/kg). Different doses of fucoidan or vehicle were administered intrathecally once daily from postoperative day (POD) 11–20. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) was measured on 1 day before operation and days 10, 20, 22, 24, 26, 28, 30 after operation. Glial activation markers such as glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1 (mac-1), inflammatory cytokines such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1 and IL-6 activation, and extracellular signalregulated protein kinase (ERK) activation in the lumbar spinal cord were determined on day 30 after operation. The results showed that fucoidan caused dosedependently attenuation of mechanical allodynia and thermal hyperalgesia. Furthermore, fucoidan could markedly inhibit neuroimmune activation characterized by glial activation, production of cytokines as well as ERK activation. The analgesic effect of intrathecal fucoidan in rats receiving SNL might partly attribute to the inhibition of neuroimmune activation associated with the maintenance of neuropathic pain. © 2014 Published by Elsevier Ireland Ltd.
Neuropathic pain develops as a consequence of a lesion or disease affecting the somatosensory pathways in the peripheral or central nervous system, and occurs in many neurological diseases. Accumulating evidence indicates that central neuroimmune activation and neuroinflammation play a pivotal role in the generation and maintenance of neuropathic pain [6,15]. After nerve injury, spinal microglia and astrocytes are activated and release pro-inflammatory cytokines, including interleukin (IL)-1, IL-6,
∗ Corresponding author. Tel.: +86 759 2396039; fax: +86 759 2396039. E-mail address: [email protected] (Y.-t. Zhao).
tumor necrosis factor-alpha (TNF-␣) and chemokines such as CCchemokine ligand (CCL) 2, CCL3, CCL5, CCL12 and fractalkine. These cytokines and chemokines elicit chronic neuroinflammation and enhance the central immune cascade, leading to neuropathic pain [10,19]. Furthermore, activated glia cells interact with neurons associated with pain transmission, and increase the excitability of these neurons, resulting in neuropathic pain [10,14]. Neuropathic pain is characterized by spontaneous pain, hyperalgesia and allodynias, affecting millions of people worldwide [24]. It is often severely debilitating and largely resistant to treatment. Currently, available therapies shown to be effective in managing neuropathic pain include opioids and tramadol, anticonvulsants, antidepressants, topical treatments (lidocaine patch, capsaicin), and ketamine. However, these treatments are either relatively
http://dx.doi.org/10.1016/j.neulet.2014.04.030 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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ineffective or accompanied by substantial side effects [16,20]. Therefore, it is urgent to clarify the molecular mechanisms underlying neuropathic pain and establish novel therapeutic strategies. Fucoidan is a sulfated polysaccharide which is one of the main constituents of brown seaweeds. Fucoidan exhibits a variety of biological activities, including antioxidant, anti-apoptosis, anticoagulant, antitumor, antiviral and anti-inflammatory activity [1,21]. However, it was still unclear whether fucoidan had beneficial effects in treating neuropathic pain. In the present study, we tested whether intrathecal fucoidan could attenuate nocifensive behavior associated with neuropathic pain. We tried to explore the underlying mechanism of the potential analgesic effect of fucoidan.
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2. Materials and methods
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2.1. Animals
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Male SPF Sprague-Dawley rats (200–250 g) were used in the present study. All animals were housed under standardized conditions in a room on a 12 h light/dark cycle with food and water available ad libitum. All experimental protocols described in this study were approved by the regulations stipulated by Guangdong Medical College Animal Care Committee which follows the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1985).
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2.2. Surgical operation
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Peripheral nerve injury was produced by SNL according to a previously described method [11]. Briefly, under chloral hydrate anesthesia (300 mg/kg, intraperitoneally, Sigma–Aldrich), a skin incision (3–4 cm) was made in the midline lumbar region (L4–S1). The L6 transverse process was identified, freed of muscular attachments, and partially removed with the help of bone ronguers. This exposed the L5 spinal nerve. The L5 was then carefully isolated and tightly ligated distal to the dorsal root ganglion (DRG) with 6–0 silk thread. The surgical procedures for the sham group were identical to that of the SNL group, except that the spinal nerves were not ligated.
2.3. Intrathecal implantation Polyethylene (PE) – 10 tube (BD, 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 L). Only those rats showing complete paralysis of both hind limbs and the tail after 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.
2.5. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) Behavioral tests were carried out between 8 and 12 am on 1 day before operation and days 10, 20, 22, 24, 26, 28, 30 after operation, respectively. Mechanical allodynia was assessed using von Frey filaments (Stoelting, Kiel, WI, USA) by experimenters blind to group assignment. The stiffness of the von Frey filaments was 2, 4, 6, 8, 10, 15, and 20 g. A positive paw withdrawal response was recorded if the animal briskly lifted the hindpaw. The mechanical withdrawal threshold (MWT) was determined by the up-down method [2]. To assess thermal sensitivity, paw-withdrawal latencies to a radiant heat stimulus were measured using the paw-flick test apparatus (IITC Life Science, Woodland Hills, CA). The thermal withdrawal latency (TWL) was recorded as the threshold of thermal sensitivity. Each hindpaw was tested five times at 5 min intervals. 2.6. Tissue harvest After finishing the behavioral tests on day 30 post operation, rats were deeply anesthetized with chloral hydrate (300 mg/kg, intraperitoneally) and then perfused intracardially with 250 ml cold saline. The ipsilateral L4–5 spinal cord tissue rostral to the injury site was removed, dissected on cold ice and removed quickly to liquid nitrogen for subsequent assays. 2.7. Real-time quantitative polymerase chain reactions Glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1 (mac-1) were markers for astrocytic and microglial activation respectively. Rats were deeply anesthetised with chloral hydrate (300 mg/kg) and sacrificed. Total RNA from the L5 spinal segments was extracted with Trizol (GIBCO/BRL Life Technologies Inc., Grand Island, NY, USA). Complementary DNA (cDNA) was synthesized with oligo (dT) 12–18 using Superscript III Reverse Transcriptase for RT-PCR (Invitrogen, Carlsbad, CA, USA). The sense primer for GFAP was 5 -GCTGGAGCAAGACAAACATTC-3 , and the antisense primer was 5 -CCCTACCCACTCCTACATCGT-3 . The sense primer for mac-1 was 5 -TGGCTGCTACTACCGTCCCT-3 , and the antisense primer was 5 -CAGGTTGGAGCCGAATAGGT-3 . The sense primer for GAPDH was 5 -CCCCCAATGTATCCGTTGTG-3 , and the antisense primer was 5 -TAGCCCAGGATGCCCTTTAGT-3 . Equal amounts of RNA (1 g) were used to prepare cDNA using the SYBR Premix Ex Taq TM (Takara, Tokyo, Japan) and analyzed with a realtime PCR detection system (Applied Biosystems, Foster City, CA, USA). All values were normalized to GAPDH expression. 2.8. Enzyme-linked immunosorbent assay Spinal production of TNF-␣, IL-1 and IL-6 was quantified by enzyme-linked immunosorbent assay (ELISA) kits for rats according to the manufacturers’ instructions (Biosource USA for IL-1 and IL-6; R&D Systems for TNF-␣). Values were expressed as picogram per milligram protein. 2.9. Western blot analysis
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2.4. Drug administration Fucoidan (Sigma, St. Louis, MO) was dissolved in 5% dimethylsulfoxide (DMSO) and then diluted with 0.9% (w/v) saline solution. Animals were divided into 5 groups for administration: shamoperation with vehicle (sham group); SNL rats received treatment with fucoidan (15 mg/kg, 50 mg/kg, 100 mg/kg) or vehicle (5% DMSO) once daily during the period of days 11–20 inclusively (n = 6 rats per treatment group).
To examine changes in p-ERK expression following SNL, all collected tissue samples were homogenized in an SDS sample buffer containing a mixture of proteinase and phosphatase inhibitors (Sigma). The protein concentrations were estimated using the bicinchoninic acid (BCA) method. Protein samples were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked in 10% skimmed milk for 1 h at room temperature and incubated with the following primary antibodies overnight at 4 ◦ C: rabbit anti-pERK (1:1000; Cell Signaling)
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and mouse anti--actin (1:3000, Sigma). The blots were further incubated for 1 h at RT with HRP-conjugated secondary antibody (anti-rabbit 1:3000, anti-mouse 1:5000; Amersham Biosciences), and washed three times in TBS for 30 min. The immune complexes were detected by the enhanced chemiluminescence (ECL) detection method (Amersham) and exposure to film. A square of the same size was drawn around each band to measure the density and the background near that band was subtracted. Target protein levels were normalized against -actin levels and expressed as relative fold changes compared to the sham group.
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2.10. Statistical analysis
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Values are expressed as means ± SEM. For behavioral data, comparisons were performed using repeated measures of analysis of variance (ANOVA). For others, comparisons were performed using ANOVA followed by Bonferroni tests. Data were analyzed using SPSS 13.0 (Chicago, IL, USA). P < 0.05 was considered significant.
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3.1. Intrathecal administration of fucoidan attenuates SNL induced mechanical allodynia and thermal hypersensitivity in a dose-dependent manner
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To investigate whether fucoidan would reverse established neuropathic pain, different doses (15, 50 and 100 mg/kg) of fucoidan were injected intrathecally once daily from postoperative day (POD) 11–20, and behavioral experiments were carried out. As shown in Fig. 1, the sham group did not display significant behavioral changes. Prominent mechanical and thermal hyperalgesic behaviors were established on day 10 after surgery as shown in the SNL-Veh group. Compared to the SNL-Veh group, treatment of fucoidan dose-dependently alleviated and reversed mechanical allodynia and thermal hyperalgesia. Especially, significant analgesic effect was observed when fucoidan was at doses of 50 and 100 mg/kg.
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3.2. The effects of fucoidan on SNL-induced spinal glial activation
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The analgesic effect of fucoidan might be associated with its inhibitory effect on microglia and astrocytes. To verify this hypothesis, we studied the expression of glial activation markers in lumbar spinal cord GFAP (Fig. 2A) and mac-1 (Fig. 2B) expression. Results showed that on day 30 after SNL, GFAP and mac-1 expression levels were markedly enhanced in SNL rats compared to the shamoperated rats (P < 0.01). Administration of various doses of fucoidan decreased spinal glial activation markers in a dose-dependent manner compared to the SNL-Veh group. Fucoidan, especially in the dose of 50 and 100 mg/kg, seems to exert higher suppressive effects. Furthermore, fucoidan suppression of spinal glial activation was accompanied by its attenuation of behavioral hyperalgesia.
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3.3. The effects of fucoidan on inflammatory cytokines
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The inhibitory effect of fucoidan on glia could lead to subsequent decreased production of inflammatory cytokines. Inflammatory cytokine (TNF-␣, IL-1, and IL-6) expression was analyzed by ELISA (Fig. 3). Results indicated that SNL surgery induced remarkable increases of inflammatory mediators including TNF-␣, IL-1 and IL-6 in the spinal dorsal horn on day 30 after surgery. However, intrathecal fucoidan significantly suppressed the elevations of these inflammatory mediators in a dose-dependent manner. Additionally, there was a significant downregulation of proinflammatory cytokines in the dose of 50 and 100 mg/kg fucoidan treatment group. Moreover, fucoidan inhibition of inflammatory
Fig. 1. The effects of intrathecal fucoidan on SNL-induced mechanical allodynia and thermal hyperalgesia. SNL injury resulted in prominent mechanical allodynia (A) and thermal hyperalgesia (B) on day 10 after surgery. The intrathecal administration of fucoidan (15, 50 or 100 mg/kg, once per day from POD 11–20) significantly blocked SNL-induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. SNL: spinal nerve ligation; POD: postoperative day. n = 6 in each group, *P < 0.05, compared to the SNL + Veh group.
cytokines expression was in line with its alleviation of hypersensitivity.
3.4. SNL-induced phosphorylation of ERK in the spinal dorsal horn is significantly inhibited by fucoidan Substantial evidence implicated that activation of mitogenactivated protein kinase (MAPK) initiates many signaling cascades and increases the synthesis of proinflammatory mediators following peripheral and central nervous injuries [7,17]. To examine whether MAPK activation was involved in the suppressive effect of fucoidan on SNL-induced inflammatory cytokines elevation, we assessed the levels of phosphorylation of ERK using western blot analysis. Our data showed that on day 30 after SNL, the phosphorylation level of ERK was significantly increased in SNL-vehicle group compared to sham-vehicle group (Fig. 4). Similar to the effect of fucoidan on inflammatory cytokines expression, the SNL-induced elevation of the phosphorylation level of ERK was also attenuated by intrathecal fucoidan.
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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Fig. 2. Expression changes of glial activation markers, GFAP and mac-1 in the lumbar spinal cord of rats on day 30 after the operation. SNL increased GFAP (A) and mac-1 (B), while fucoidan dose-dependently down-regulated the markers mentioned above. n = 6 in each group, *, **: P < 0.05, P < 0.01 compared to the sham + Veh group. #, ##: P < 0.05, P < 0.01 compared to the SNL + Veh group.
Fig. 3. The effects of intrathecal fucoidan on the proinflammatory associated cytokines. The protein levels of the proinflammatory cytokines TNF-␣ (A), IL-1 (B), and IL-6 (C) were determined in the spinal dorsal horn in rats that received different treatments using ELISA. Fucoidan significantly prevents and reverses SNL-induced increases of these inflammatory mediators. n = 6 in each group, *, **: P < 0.05, P < 0.01 compared to the sham + Veh group. #, ##: P < 0.05, P < 0.01 compared to the SNL + vehicle group. The levels of proinflammatory cytokines are expressed as relative fold changes compared to the Sham + Veh group.
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4. Discussion In the present study, we observed that repeated administration of fucoidan dose-dependently attenuates the existing mechanical allodynia and thermal hyperalgesia induced by SNL. Subsequently, we investigated the mechanisms that underlie the analgesic effect of fucoidan. We found that intrathecal fucoidan could block the maintenance of neuropathic pain. Furthermore, fucoidan significantly inhibits spinal astrocytic and microglial activation, proinflammatory mediators production and MAPK activation. Our results suggest that
fucoidan may have therapeutic potential in attenuating neuropathic pain, possibly through inhibiting spinal neuroimmune activation. Activation of MAPK has been implicated in a number of signaling events that are important for the initiation and maintenance of neuropathic pain. In the SNL neuropathic pain model, it has been reported that MAPK including ERK, p-38 and JNK are activated in microglia and or astrocyte [8,10,22,23]. We also observed ERK activation in the current study. After repeated intrathecal fucoidan, the phosphorylation level of ERK declined. A previous study has shown that partial sciatic nerve ligation (PSNL) increased p38-MAPK
Please cite this article in press as: C. Hu, et al., Fucoidan attenuates the existing allodynia and hyperalgesia in a rat model of neuropathic pain, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.030
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Fig. 4. The effects of intrathecal fucoidan on SNL-Induced ERK activation. The expression of phosphorylated (p) ERK (A and B) was revealed by Western blot analysis. On day 30 after SNL, the phosphorylation level of ERK is significantly increased in SNL vehicle rats. After treatment with fucoidan (100 mg/kg) from SNL day 11 to day 20, the phosphorylation level is remarkably decreased. n = 6 in each group, *P < 0.05 compared to the sham group. #P < 0.05 compared to the SNL + vehicle group. The results are expressed as relative fold changes compared to the Sham + Veh group.
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activation [13]. However, clonidine suppressed activation of p38 MARK in sensory neurons after nerve injury [4]. As inhibition of the MAPK signaling pathway could contribute to inflammation response, the suppression of MAPK activation by fucoidan further added to its role on neuropathic pain and the associated neuroimmune activation. Neuroimmune activation including glial activation and inflammatory cytokine release contributes to neuropathic pain. After peripheral and central nervous injuries, astrocytes and microglia are activated. Then these glial cells release inflammatory cytokines, which amplify neuronal excitation and pain signaling. Therefore, drugs that suppress glial activation and inflammatory cytokines production may prevent and reverse neuropathic pain. In this study, we observed that fucoidan suppressed spinal glial activation and proinflammatory cytokines production. The inhibitory effects of fucoidan on neuroinflammation are in agreement with several other studies on the effects of fucoidan on inflammation diseases. It has been shown that fucoidan significantly inhibited lipopolysaccharide (LPS)-activated microglial inflammatory responses [18]. Moreover, it inhibited the expression of some cytokine/chemokine in LPS accelerated cerebral ischemic injury rats [9]. In addition, fucoidan could ameliorate the learning and memory abilities in A-induced Alzheimer’s disease rats [5]. Fucoidan could also attenuate ischemia-reperfusion (I/R)-induced myocardial damage and prevent the loss of dopaminergic neurons in a LPS-induced animal model of Parkinson’s disease [3,12]. These data suggest that fucoidan has anti-inflammatory activities and neuroprotective effects. It could be considered for use as a potential treatment for inflammatory and neurodegenerative disorders. Acknowledgments
This work was supported by grants from the Doctoral ProQ2 gram of Guangdong Medical College, China (XB1021), the Science 286 and Technology Project Foundation of Zhanjiang City, China 287 (2010C3104009), the Medical Scientific Research Foundation of 288 Guangdong Province, China (B2010234) and the Breeding Project 289 of the Education Department of Guangdong Province, China 290 (LYM10084). We declare no conflicts of interests. 291 285
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