NF-κB Mediated In Thermal Hyperalgesia And Molecular Mechanisms Following Chronic Compression Of The Dorsal Root Ganglion In Rats

BackgroundMechanical compression of the dorsal root ganglion (DRG) and the nerve root by spinal canal stenosis or disc herniation is one of the primary causes of radicular pain syndrome in humans. Chronic compression of DRG (CCD) in animals, a typical model of neuropathic pain, results in spontaneous pain, mechanical allodynia and thermal hyperalgesia. Peripheral nerve injury induces neuropathic pain by upregulating a complicated network of molecules in DRG and spinal cord, including the proinflammatory cytokine tumor necrosis factor (TNF)-a, interleukin (IL)-6and nuclear factor-kappa B (NF-κB). Neurotransmitter and second messenger signals, including nitric oxide (NO), cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG), have been shown to participate in maintaining neuronal hyperexcitability and behavioral hyperalgesia in CCD treatment. Moreover, several types of ion channels such as voltage-gated Na+and K+, hyperpolarization-activated cation current, and transient receptor potential vanilloid4(TRPV4) may contribute to the transmission of nociceptive stimuli in CCD neurons. NO has been shown to be a key mediator of nociceptive activity in animal pain models and involved in the maintenance of pathological pain. TRPV4synthetic activator,4a-PDD, and hypoosmotic stimulation could induce NO production in outer hair cells of the guinea pig cochlea, while NO production is inhibited by ruthenium red, an inhibitor of TRPV4. Our recent study indicated that TRPV4enhances pain-related behavior through a NO-cGMP-PKG cascade that may occur within the DRG in CCD rat. However, up to date, the detailed mechanisms between ion channels (TRPV4) and neurotransmitter (NO) in behavioral hyperalgesia remain unclear in the CCD model of neuropathic pain in rats.NF-κB consists of multi-submit proteins of Rel family (p50, p52, p65, c-Rel, and RelB). Under normal conditions or in resting cells, NF-κB is located in the cytoplasm and bound with inhibitory proteins, generically called inhibitory factor B (I-κB). When the cell is activated, proteins are degraded via the ubiquitin-proteasome pathway, leading to the release of the active form of NF-κB which translocates to the nuclear, where it regulates the expression of multiple genes involved neuropathic pain. Recently, activation of NF-κB has been shown to be involved in the transmission and processing of nociceptive information in the DRG and spinal cord. The analgesic effects of both low and high frequency electroacupuncture significantly decreased in NF-κB knockout mice. Further, intrathecal administration of NF-κB inhibitors, PDTC (pyrrolidine dithiocarbamate) and SN50, prior to glycoprotein partially attenuated glycoprotein-induced allodynia and pretreatment with NF-κB antisense oligodeoxynucleotides (ODNs) attenuated mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. These evidence suggests that NF-κB may be involved in the neuropathic pain. Therefore, we hypothesized that NF-κB might be involved in TRPV4-NO in thermal hyperalgesia in the CCD model. In order to test this hypothesis, we investigated the effects of PDTC and BAY, two specific NF-κB inhibitors, on paw withdrawal latency in the DRG in CCD model.ObjectiveTo investigate the effects of the two specific NF-κB inhibitors, PDTC and BAY, on paw withdrawal latency in the DRG in CCD model.Methods1. Establish CCD modelMale Wistar rats (n=80), weighing150-180g, were purchased from the Animal Center of Shandong University and were housed in a controlled environment (50-60%relative humidity,12h light/dark cycle, room temperature23±2℃), with free access to food and water for at least7days before surgery. Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg ip), the transverse process and intervertebral foramina of L4and L5were exposed unilaterally as previously described. A stainless steel L-shaped rod (0.63-mm diameter and4-mm length) was inserted into each foramen, one at L4and the other at the L5level, to compress the DRG. The incision site was sutured in layers and penicillin was injected to prevent infection. The sham group underwent the same surgical procedure as described, but without the insertion of the rods. The animals did not show any autotomy and any loss of sensation following the surgery. All efforts were made to minimize animal suffering and the number of animals used. The study was approved by the Chinese Institutional Care Committee for the use of animals and was performed in accordance with the Helsinki declaration.2. Chemicals and treatment4a-phorbol12,13-didecanoate (4α-PDD, TRPV4synthetic activator) and pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), were purchased from Sigma Company (St Louis, MO), BAY11-7082(BAY, an NF-κB inhibitor) was purchased from Beyotime Institute Biotechnology of (Shanghai, China). All chemicals were dissolved in saline. PDTC and BAY were respectively reconstituted in nuclease-free0.9%NaCl (10μg/μl) and administered into the spinal intrathecal space once a day for7days until the animals were sacrificed.3. Behavioral testingThermal hyperalgesia was assessed using the paw withdrawal latency (PWL) to radiant heat according to the protocol of Villetti and colleagues. Rats were placed in clean cages with a6-mm thick glass floor and acclimate to their surroundings for15-20min before testing. A high intensity light beam beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw through the glass plate. The nociceptive endpoints in the radiant heat test were the characteristic lifting or withdrawal of the hind paw, while the time to the endpoint was defined as the paw withdrawal latency. To avoid tissue damaged, the cut-off time was set at20s. There were three trials per rat with5min intervals between trials. The rats were tested on each of2successive days before surgery. Postoperative tests were performed2h before chemicals treatment on the7th day after surgery. Additional tests were conducted1,2,4,8, and24h after injection of chemicals or saline into the subarachnoid space on the midline between the L4and L5vertebrae on the7th day after surgery.4. Data analysisA two-way repeated measures analysis of variance (RM ANOVA) was used to compare the differences in PWL over time between several treatment and control groups.Results1. Effects of PDTC (a specific inhibitor of NF-κB) on CCD-induced thermal hyperalgesia in ratsThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=245.054, P<0.001). The intrathecal administration of the inhibitor of NF-κB PDTC in concentration range of10-1-10-2M produced a significant dose-dependent reduction of the PWL when compared with saline group, but PDTC in a concentration of the10-3M had no effects on thermal hyperalgesia (n=8, P>0.05). The significant reduction of the thermal hyperalgesia was observed at1h (P<0.05), peaked at4h (P<0.05), and lasted for about8h post-injection (P<0.05). There were no significant differences between groups in CCD rats at24h after administration of PDTC (n=8, all P>0.05).2. Effects of BAY (a specific inhibitor of NF-κB) on CCD-induced thermal hyperalgesia in ratsThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=253.816, P<0.001). Intrathecal pretreatment with BAY (100-50μM) significantly and dose-dependently inhibited CCD-induced thermal hyperalgesia when compared with saline group. The significant decrease of the thermal hyperalgesia was observed from1to8h (P<0.05), peaked at4h (P<0.05). The intrathecal administration of BAY in concentration of (25μM) had no effects on thermal hyperalgesia when compared to saline group (n=8, P>0.05).3. Effects of4a-PDD on the PDTC-induced suppression of thermal hyperalgesia in rats4α-PDD (the TRPV4agonist,1nm) attenuated the suppressive effects of PDTC (10-1M) on CCD-induced thermal hyperalgesia when compared to PDTC (10-1M) alone (n=8, P<0.05).4. Effects of4α-PDD on the BAY-induced suppression of thermal hyperalgesia in rats4α-PDD (the TRPV4agonist,1nm) attenuated the suppressive effects of BAY (100μM) on CCD-induced thermal hyperalgesia when compared to BAY (100μM) alone (n=8, P<0.05).ConclusionsThis study demonstrates that NF-κB could be involved in TRPV4-NO pathway in CCD-induced thermal hyperalgesia following chronic compression of the dorsal root ganglion in rat. BackgroundNitric oxide (NO) as a mediator of pathological nociception, acting as an inter-and intracellular messenger molecule in the peripheral and central nervous system, plays a pivotal role in the development and maintenance of hyperalgesia and neuropathic pain. Three isoforms of NO synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), mediate NO synthesis from L-arginine in the presence of oxygen. These NOS isoforms'expression can be upregulated in nervous tissues under pathological conditions, suggesting that the pathophysiologic functions of NO in the nervous system may be regulated by altering expression and activity of NOS isoforms. Neuronal NOS is expressed in the neurons and produces predominantly NO in neuronal tissues. The contribution of nNOS-synthesized NO to nociceptive processing has been characterized in several neuropathic pain models. In recent years iNOS has become of significant interest in the pathophysiology of neuropathic pain. iNOS expressed in neuronal tissues is thought to be involved in mechanisms of hyperalgesia with some authors showing a specific role in the pain hypersensitivity associated with models of spinal cord injury. Increased nNOS and iNOS expression in neuronal tissues have also been observed in the chronic constriction injury (CCI) model and spinal nerve ligation, suggesting their role in the development of neuropathic pain, lines of evidence have demonstrated that nNOS and iNOS contribute to NO production involved in the maintenance of behavioural hyperalgesia developing after spinal nerve transection and sciatic nerve chronic constriction injury. However, its contribution to behavioural hypersensitivity in other animal models of neuropathic pain still remains unclear.NF-κB consists of multi-submit proteins of Rel family (p50, p52, p65, c-Rel, and RelB). Under normal conditions or in resting cells, NF-κB is located in the cytoplasm and bound with inhibitory proteins, generically called inhibitory factor B (I-κB). When the cell is activated, proteins are degraded via the ubiquitin-proteasome pathway, leading to the release of the active form of NF-κB which translocates to the nuclear, where it regulates the expression of multiple genes involved neuropathic pain. The NF-κB activation process has two pathways. First, it is the positive feedback pathway in the extracellular, this is an important mechanism for amplification of inflammatory reactions pro-inflammatory mediators TNF-alpha, IL-1and reactive oxygen medium (ROI) is a strong inducer of NF-kappa B. Pro-inflammatory mediators in the activation of NF-κB belongs to the positive feedback regulation, the induction of NF-κB activation can enhance TNF-a and IL-1β gene transcription, increased production and release, and then re-activation of NF-kappa B; also allows the increase in IL-6and IL-8production and release, resulting in further amplification of the initial inflammatory signal. Secondly, NF-kappa B activation start at the gene transcription of inflammatory mediators such as IκB-α and p105gene transcription were also upregulated. IκB gene the promoter region containing the κB sequence and kappa B sequence degradation, and thus can be combined with NF-κB, or even to enter the nucleus and activation of NF-κB binding, inhibition of NF-κ B and DNA binding site.Recently, activation of NF-κB has been shown to be involved in the transmission and processing of nociceptive information in the DRG and spinal cord. The analgesic effects of both low and high frequency electroacupuncture significantly decreased in NF-κB knockout mice. Further, intrathecal administration of NF-κB inhibitors, PDTC (pyrrolidine dithiocarbamate) and SN50, prior to glycoprotein partially attenuated glycoprotein-induced allodynia and pretreatment with NF-κB antisense oligodeoxynucleotides (ODNs) attenuated mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. These evidence suggests that NF-κB may be involved in the neuropathic pain. Therefore, we hypothesized that NF-κB might be involved in TRPV4-NO in thermal hyperalgesia in the CCD model. In order to test this hypothesis, we investigated the effects of PDTC and BAY, two specific NF-κB inhibitors, on paw withdrawal latency in the DRG in CCD model.Objective1. To detect the NO (nitric oxide) content in rats in the CCD model. 2. To investigate the effects of CCD on the expression levels of mRNA and protein of NF-κB and I-κB.3. To determine the role of NF-κB in CCD-induced TRPV4-NO pathway.Methods1. Establish CCD modelMale Wistar rats (n=80), weighing150-180g, were purchased from the Animal Center of Shandong University and were housed in a controlled environment (50-60%relative humidity,12h light/dark cycle, room temperature23±2℃), with free access to food and water for at least7days before surgery. Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg ip), the transverse process and intervertebral foramina of L4and L5were exposed unilaterally as previously described. A stainless steel L-shaped rod (0.63-mm diameter and4-mm length) was inserted into each foramen, one at L4and the other at the L5level, to compress the DRG. The incision site was sutured in layers and penicillin was injected to prevent infection. The sham group underwent the same surgical procedure as described, but without the insertion of the rods. The animals did not show any autotomy and any loss of sensation following the surgery. All efforts were made to minimize animal suffering and the number of animals used.2. Measurement of NO content in DRGThe level of nitrite as a measure of NO production in DRG was determined with modified Griess reagent. A nitrite detection kit (Beyotime Biotech Inc., Jiangsu, People's Republic of China) was used according to instructions provided by the manufacturer.3. Nuclear and cytoplasmic protein extraction Frozen tissues were homogenized in cytoplasmic extraction reagent A CERA (0.2ml)(BioTeke Corporation, Beijing, China). After vortexing for10seconds, the homogenates were centrifuged at15,000rpm for10minutes at4℃, the supernatant containing cytoplasmic proteins were collected and stored at-80℃. After centrifugation at15,000rpm for10min at4℃, the supernatant was collected as nuclear extracts and stored at-80℃.4. Western blotSix DRGs from3rats were pooled to extract protein as one sample and were quickly frozen in liquid nitrogen, then stored at-80℃for further examination. The amount of protein in the cytoplasmic or nuclear extracts was quantified by Protein Quantitative Analysis kit (k3001-BCA; Shenergy Biocolor, Shanghai, China). The body homogenates were electrophoresed and transferred to nitrocellulose membranes. Membranes were blocked for1h at room temperature in blocking buffer, washed in TTBS (0.1%Tween20,50mM Tris, and150mM NaCl), and incubated overnight with rabbit anti-NF-KB antibody (1:500, sc8008; Santa Cruz) or rabbit anti-IκB antibody (1:800, sc371; Santa Cruz). After washing three times, membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000).5. Real-time PCRThe mRNA levels of NF-κB,I-Kb and nNOS in the DRG in each group were quantified by real-time reverse-transcriptase polymerase chain reaction (RT PCR) using SYBR Green technology. 7. ImmunohistochemistryAt7days post-surgery rats were anesthetized with isoflurane and then transcardially perfused with4%paraformaldehyde in phosphate-buffered saline (PBS, pH7.4);the DRGs was immediately removed and post-fixed in4%paraformaldehyde (PFA) for7-8h, then the tissues were treated with dehydration, embedded in paraffin and cut into serial paraffin sections (5μm). The paraffin-embedded sections were heated for2h at120℃, deparaffinized in xylene, and rehydrated through graded ethanol at room temperature. After three rinses in PBS, microwave accentuation was used for10minutes; then the sections were washed in PBS,3%H2O2to eliminate endogenous peroxydase, and blocked with10%normal goat serum; then sections were incubated overnight at4℃with primary antibodies, anti-SP polyclonal antibody (1:100, Santa Cruz) or anti-CGRP polyclonal antibody (1:100, Santa Cruz). After the sections were washed, they were incubated with anti-rabbit IgG peroxidase conjugate (1:300, Zhongshan Gold Bridge, Beijing, China) for1h at room temperature. After several rinses, peroxidase was revealed by a3,3'-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Gold Bridge, Beijing, China). Finally, the sections were weakly counterstained with hematoxylin. In negative controls, the sections were incubated with PBS instead of primary antibody. All images were captured and analyzed by use of a color image analysis system composed of a video camera (Olympus DP71, Olympus Co., Japan), a light microscope (Olympus BX51, Olympus Co.), and Image-Pro Plus5.0software (Media Cybernetics Inc., USA).Results 1. Effects of PDTC and BAY (two specific inhibitors of NF-κB) on CCD-induced thermal hyperalgesia and the NO content in the DRGThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=245.054, P<0.001). The intrathecal administration of the inhibitor of NF-κB PDTC in concentration range of10-1-10-2M produced a significant dose-dependent reduction of the PWL when compared with saline group, but PDTC in a concentration of the10-3M had no effects on thermal hyperalgesia (n=8, P>0.05). The significant reduction of the thermal hyperalgesia was observed at1h (P<0.05), peaked at4h (P<0.05),and lasted for about8h post-injection (P<0.05). There were no significant differences between groups in CCD rats at24h after administration of PDTC (n=8, all P>0.05). Intrathecal injection of PDTC (10-1M) significantly decreased the content of NO metabolites nitrite when compared to saline (n=8, P<0.05) in DRG in CCD rats. The level of NO in DRG showed the decrease1h post-injection significantly (P<0.05) and reached the minimum4h post-injection (P <0.05), followed by a gradual recovery (P<0.05), and the NO content was not significantly different from the saline group at24h post-injection (n=8, P>0.05). The reduction of NO content in DRG was negatively associated with the increase in PWL (r=-0.966, P<0.05) post-operation.The PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=253.816, P<0.001). Intrathecal pretreatment with BAY (100-50μM) significantly and dose-dependently inhibited CCD-induced thermal hyperalgesia when compared with saline group. The significant decrease of the thermal hyperalgesia was observed from1to8h (P<0.05), peaked at4h (P<0.05). The intrathecal administration of BAY in concentration of (25μM) had no effects on thermal hyperalgesia when compared to saline group (n=8, P>0.05). Moreover, the level of NO in the DRG of CCD-treated rats was significantly suppressed by intrathecal pretreatment with BAY (100μM). The level of NO began to decrease1h post-injection (P<0.05) and reached the minimum4h post-injection (P<0.05), followed by a gradual recovery (P<0.05), and the NO content was not significantly different from the saline group at24h post-injection (n=8,P>0.05). The reduction of NO content in DRG was negatively associated with the increase in PWL (r=-0.952, P<0.05) post-operation.2. Effects of4a-PDD on the two specific inhibitors of NF-κB (PDTC and BAY) induced suppression of thermal hyperalgesia and NO content in the DRG4a-PDD (the TRPV4agonist, lnm) attenuated the suppressive effects of PDTC (10-1M) on CCD-induced thermal hyperalgesia when compared to PDTC (10-1M) alone (n=8, P<0.05). Moreover, the decrease in the content of NO induced by intrathecal injection of PDTC (10-1M) was also attenuated by pretreatment of4α-PDD (1nm)1h post-injection (n=8, P<0.05).4a-PDD (the TRPV4agonist, lnm) attenuated the suppressive effects of BAY (100μM) on CCD-induced thermal hyperalgesia when compared to BAY (100μM) alone (n=8, P<0.05). Moreover, the reduced content of NO induced by intrathecal injection of BAY (100μM) was also attenuated by pretreatment with4a-PDD (1nm)1h post-injection (n=8, P<0.05).3. Western blot of nuclear NF-κB,cytoplasmic I-κB and nNOS expression in the DRGThe effects of PDTC on the inhibition of NF-κB expression were confirmed at the protein level by western blot. Compared with the sham group, CCD rats exhibited significantly higher levels of NF-κB expression (P<0.05). The expression of NF-κB was markedly attenuated after intrathecal injection of PDTC (P<0.05). CCD rats showed significantly lower levels of cytoplasmic I-κB expression when compared with the sham group (P<0.05). The reduction of I-κB expression was reversed after intrathecal injection of PDTC (P<0.05). In addition, compared with the sham group, CCD rats showed significantly higher nNOS (P<0.05). The expression of nNOS was markedly attenuated after intrathecal injection of PDTC (P<0.05).4. Effects of CCD on gene expression of nuclear NF-κB,cytoplasmic I-κB and nNOS in the DRGThe effects of PDTC on the inhibition of NF-κB mRNA expression were confirmed at the protein level by RT-PCR. Compared with the sham group, CCD rats exhibited significantly higher levels of NF-κB mRNA expression (P<0.05). The expression of NF-κB mRNA was markedly attenuated after intrathecal injection of PDTC (P<0.05). CCD rats showed significantly lower levels of cytoplasmic I-κB mRNA expression when compared with the sham group (P<0.05). The reduction of I-κB mRNA expression was reversed after intrathecal injection of PDTC (P<0.05). In addition, compared with the sham group, CCD rats showed significantly higher nNOS mRNA (P<0.05). The expression of nNOS mRNA was markedly attenuated after intrathecal injection of PDTC (P<0.05).5. Effects of CCD on positive cell of NF-κB and nNOS in the DRGThe positive cell of NF-κB and nNOS in DRG were determined by immunohistochemistry. Our data showed that there was a significant increase in positive cell of NF-κB and nNOS in DRG compared with that in sham group rats (all P<0.05). The positive cell of NF-κB and nNOS in DRG were decreased intrathecal injection of PDTC as compared to CCD groups (all P<0.05).ConclusionsNF-κB might induce nitric oxide synthase, which mediated in TRPV4-NO pathway in CCD-induced molecular mechanisms.