The Journal of Pain
Volume 12, Issue 1 , Pages 141-152, January 2011

Topical Application of Compound Ibuprofen Suppresses Pain by Inhibiting Sensory Neuron Hyperexcitability and Neuroinflammation in a Rat Model of Intervertebral Foramen Inflammation

Department of Neurobiology, Parker Research Institute, Dallas, Texas

Received 30 March 2010; received in revised form 4 June 2010; accepted 15 June 2010. published online 27 August 2010.

Article Outline

Abstract 

There is lack of evidence that topical application of an anti-inflammatory reagent could reduce pain due to intervertebral foramen (IVF) inflammation (IVFI). We investigated analgesic effects and underlying mechanisms of topical application of a compound ibuprofen cream (CIC) onto the surface of back skin covering the inflamed L5 IVF in a rat model. Repetitive CIC treatment (∼.54 g each treatment daily for 5 consecutive days) significantly reduces severity and duration of IVFI-induced thermal hyperalgesia and mechanical allodynia by 80 to 100% and 50 to 66%, respectively. Electrophysiological studies and Western blot analysis demonstrated that CIC treatment significantly inhibited hyperexcitability of the inflamed dorsal root ganglion (DRG) neurons and upregulation of Nav1.7 and Nav1.8 protein, respectively. Pathological manifestations of the inflamed DRG were also markedly improved following CIC treatment. Further, in the inflamed DRGs, phosphorylation and expression of transcription factor NF-κB and pro-inflammatory enzyme cyclooxygenase-2 (COX-2) were significantly increased, while a cytokine IL-1β level was increased. IVFI-induced upregulation of these molecules was significantly inhibited by CIC treatment. This study provides evidence that an anti-inflammatory reagent can be used topically to suppress pain due to IVFI and/or DRG inflammation through inhibition of sensory neuron hyperexcitability and the immune and inflammatory responses.

Perspective

This study suggests a convenient and safe clinical intervention for treating pain due to intervertebral foramen inflammation and similar syndromes.

Key words: Dorsal root ganglion, sodium channel, NF-κB, COX-2, IL-1β

 

Lumbar intervertebral foramen (IVF) inflammation (IVFI) and the within dorsal root ganglion (DRG) neuron inflammation play critical roles in the pathogenesis of neuropathic and inflammatory pain. This process can produce injury or disease to the structures and tissues within and/or adjacent to the IVF.4, 7, 37 Following inflammation or nerve injury or DRG compression, the chemical factors such as cytokines, nerve growth factors, inflammatory mediators, and other substances release and activate and/or change the properties of DRG neurons and the spinal dorsal horn neurons and contribute to hyperalgesia.1, 5, 22, 39, 40, 42, 43, 44 Nerve-injured or DRG-compressed sensory neurons, in vitro, exhibit enhanced responses to inflammatory mediators.22, 40 We have recently reported that in vivo delivery of inflammatory soup (IS) directly into the rat lumbar IVF results in thermal hyperalgesia, mechanical allodynia, and DRG neuron hyperexcitability.41, 45 This model may be useful for understanding mechanisms and examining treatment effects in some forms of low back pain resulting more directly from DRG neuron inflammation and inflammation within the local IVF.2, 27, 41, 45 Although many anti-inflammatory reagents have been used topically to attenuate pain syndromes in similar disorders, there is no evidence demonstrating that topical application of an anti-inflammatory reagent on back skin covering the inflamed IVF can alleviate pain due to IVFI and/or neuroinflammation. Further, it is unknown whether this action would be related to modulation of the reagent on the DRG neuron excitability and the voltage-gated sodium channels (VGSCs), which are necessary for electrogenesis and nerve impulse conduction and considered to be important in modulation of the DRG neuron excitability and behavioral hyperalgesia;6, 17, 32, 46 whether the reagent would inhibit the inflammatory response by regulating the transcription factor NF-κB, which is a central coordinator of immune and inflammatory responses, and whether expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and induction of cytokines such as IL-1β that play important roles in chronic inflammation, neurodegeneration, and neural synaptic plasticity1, 19, 23, 28, 29 would, therefore, be changed by the anti-inflammatory reagent.

Compound ibuprofen cream (CIC) is a topically applied formula consisting of ibuprofen, glucosamine, chondroitin, methylsulfonylmethane, and bromelain, clinically formulated with the proprietary liposomal dermal delivery matrix. These components are known as anti-inflammatory and topical pain relievers. Synergistic effects of this formula target the source of pain and inflammation, avoiding negative side effects including stomach upset associated with over-the-counter medications taken orally. However, there is lack of experimental evidence that supports the efficacy of this CIC in treating pain, and neural mechanisms underlying its analgesic effect remains unknown. The present study was undertaken first to document potential analgesic effect of topical application of CIC on the back skin covering the segments of the inflamed IVF and DRG. To further understand the underlying mechanisms, we then investigated alterations of the DRG neuron excitability, sodium channels Nav1.7 and Nav1.8, pathological and molecular manifestations of the DRG inflammation, activity of the transcription factor NF-κB, expression of the proinflammatory enzyme COX-2, and the cytokine IL-1β level in DRGs in IVFI rats with or without CIC treatment.

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Methods 

Animals 

All experimental procedures were conducted in accordance with the recommendations of the International Association for the Study of Pain (IASP) and the National Institute of Health Guide for the Care and Use of Laboratory Animals. The procedures were reviewed and approved by the Institutional Animal Care Committee. Adult male Sprague-Dawley rats (200–250 g weight at start of the experiment, n = 170; purchased from Charles River Laboratories, Wilmington, MA) were used in this study. They were housed in groups of 4 to 5 in 40- × 60- × 30-cm plastic cages with soft bedding and free access to food and water under a 12-hour day/night cycle. The rats were kept 3 to 5 days under these conditions before and up to 35 days for some animals after surgery. All surgeries were done under anesthesia induced by sodium pentobarbital (40 mg/kg, ip, 1 dose).

IVFI 

IVFI was produced by in vivo delivery of IS directly into the lumbar IVF at L5 in 126 rats. The procedure was the same as that which we have previously described.41, 45 In brief, each rat was anesthetized and a midline incision was made between L4-L6. On the left side, the paraspinal muscles were separated from the mammillary process and the transverse process and the L5 IVF exposed. A fine, sharp, stainless steel needle, .4 mm in diameter, attached with a micro syringe, was inserted approximately 2 mm into the IVF at a rostral direction at an angle of ∼30 to 40° to the dorsal midline and –10 to –15° to the vertebral horizontal line. The insertion was guided by the mammillary process and the transverse process. As the needle was moved over the ganglion and/or the nerve, the ipsilateral hind leg muscles typically exhibited 1 or 2 slight twitches. Then, the IS (15 μl) containing Bradykinin, 5-HT, Histamine, and prostaglandin each in 10−5 M, at pH 7.35 or other vehicle was slowly injected (manually guided) into the IVF. After the needle was withdrawn, the muscle and skin layers were sutured. Of the 126 rats, 52 were used for behavioral testing; 10, 54, and 10 received electrophysiological, Immunoblot/ELISA, and pathological assessment, respectively. In a separate group of 44 rats, the surgical procedure was identical to that described in IVFI but without needle stick and injection of IS used as sham control. Of these 44 rats, 16 were used for behavioral testing; 5, 18, and 5 received electrophysiological, Immunoblot/ELISA, and pathological assessment, respectively.

Behavioral Observations and Tests 

The rats were tested on each of 2 successive days prior to surgery. After surgery, the animals were inspected every 1 or 2 days during the first 14 postoperative days and at weekly intervals thereafter until 35 postoperative days. For general observation, the rats were placed on a table and notes were made on the animal's gait and the posture of each hindpaw and the conditions of the hindpaw skin.

The presence of thermal hyperalgesia was determined by measuring foot withdrawal latency to heat stimulation.42, 44, 53 Each rat was placed in a box (22 × 12 × 12 cm) containing a smooth glass floor. The temperature of the glass floor was measured and maintained at 26 ± .5°C. A heat source (IITC Model 336 Analgesia Meter, Series 8; Life Science Products, Frederick, CA) was moved beneath a portion of the hindpaw that was flush against the glass and a thermal stimulus was delivered to that site. The stimulus shut off automatically when the hindpaw moved (or after 20 seconds to prevent tissue damage). The intensity of the heat stimulus was maintained at a constant throughout all experiments. The elicited paw movements were at a latency of approximately 9 to 12 seconds in control rats. Thermal stimuli were delivered 4 times to each hind paw at 5- to 6-minute intervals. For assessment of thermal hyperalgesia, the withdrawal latency on the contralateral side was subtracted from those on the experimental side and the result was expressed as a difference score.

The presence of mechanical allodynia was determined by measuring foot withdrawal threshold to mechanical indentation of the plantar surface of each hindpaw with Von Frey filaments.42, 44, 53 The filaments, capable of exerting forces of 10, 20, 40, 60, 80, and 120 mN but each having the same tip diameter of .1 mm, were applied to 10 designated loci distributed over the plantar surface of the foot. During each test, the rat was placed in a transparent plastic cage with a floor of wire with 1- × 1-cm openings. The cage was elevated so that stimulation could be applied to each hind foot from beneath the rat. Each filament was applied alternately to each foot and to each locus. The filaments were applied in order of ascending force. The duration of each stimulus was 1 second and the interstimulus interval was approximately 10 to 20 seconds. The incidence of foot withdrawal was expressed as a percentage of the 10 applications of each stimulus as a function of force. The threshold was defined as the force corresponding to a 50% withdrawal, as determined by linear interpolation. To reduce preexisting differences among individuals in mechanical responsiveness, the withdrawal thresholds were also normalized by subtracting each value on the treated side from the corresponding value on the contralateral side and the results were expressed as difference scores. The testing schedule was the same as that in the thermal test.

Excised, Intact DRG Neuron Preparation 

In vitro L5 DRG preparations were made from 20 rats (5 rats in each of these groups: IVFI, IVFI+CIC, IVFI+Vehicle, and sham control) during 14 to 15 postoperative days. Under anesthesia, the sciatic nerve was isolated from surrounding tissue, transected at the midthigh level, and its proximal portion traced to the ganglia. A laminectomy was then performed and the L5 DRG and its dorsal roots were identified. Oxygenated artificial cerebrospinal fluid (ACSF), consisting of (in mM) 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 10 dextrose, 1.2 MgCl2, and CaCl2 (pH = 7.3), was dripped periodically onto the surface of the ganglion during the surgical procedure to prevent drying and hypoxia. The ganglion was removed from the rat and placed in a 35-mm petri dish filled with oxygenated ACSF. Under the dissecting microscope, the perineurium and epineurium were peeled away from the ganglion with fine forceps and the attached peripheral nerve and dorsal roots transected adjacent to the ganglion. The ganglion was then placed in the recording chamber and mounted on the stage of an upright microscope (BX50-WI; Olympus, Japan). A U-shaped stainless steel rod with 4 pieces of silver wire crossed from 1 side to the other was used to gently hold the ganglion in place within the recording chamber. The DRG was perfused continuously with oxygenated ACSF at room temperature.

Intracellular Electrophysiological Recordings 

Intracellular, electrophysiological recordings were made from the DRG somata using conventional bridge-balance techniques (Axoclamp-2B; Axon Instruments, Foster City, CA) and analyzed with PCLAMP-8 under Windows 98 (Axon Instruments). DRG cells were visualized under differential interference contrast in the microscope and the cell soma was classified visually by the diameter of its soma as small (≤30 μm), medium (31–49 μm), or large (≥50 μm). The small cells are nociceptive cells and are somata of the unmyelinated C-fibers that convey nociceptive information from peripheral terminals to the spinal cord or higher levels of the central nervous system. The medium cells match Aδ-fibers and mainly convey sharp and fast pain. The large cells match the big Aβ-fibers, and primarily convey non-nociceptive information, such as touch, light pressure, etc.38 Glass microelectrodes were fabricated with a Flaming/Brown micropipette puller (Model P-97/PC; Sutter Instruments, Novato, CA) and were filled with 2M potassium acetate (pH = 7.2). Satisfactory recordings were obtained with electrodes having DC resistances ranging from 20 to 60 MΩ.

For evaluating the DRG neuron excitability, we examined resting membrane potential (Vm), the action potential (AP) current threshold, and the repetitive discharge characteristics of the cells evoked by depolarizing current. The Vm was taken 2 to 3 minutes after a stable recording was first obtained. Depolarizing currents of .05 to 2 nA (50-ms duration) were delivered in increments of .05 nA (for small cells) or .1 to .2 nA (for medium and large cells) until an AP was evoked. AP current threshold was defined as the minimum current at 50-ms duration required to evoke an AP. Repetitive discharge of each neuron was measured by counting the spikes evoked by intracellular injection of standardized depolarizing currents at 2.5 × threshold strength (×1000 ms). Discharge patterns of DRG neurons were also classified into 2 types: 1) neurons firing either 1 or 2 APs; and 2) neurons firing >2 APs.42, 44

Western Blot Analysis 

Western blot analysis was used to detect protein expression levels in the DRGs. The L5 ganglions were isolated and stored at –80°C. Each sample consisted of 2 ganglia from 2 rats. A total of 3 samples from 6 rats were used for each group. Sequential precipitation procedures were used on the tissue samples that were lysed in ice-cold (4°C) lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, .5% deoxycholate acid, .1% SDS, 2 mM EDTA, 10 μg/mL aprotinin, 1 μg/mL leupeptin, 10 μg/mL pepstatin, .4 mM 4-[2-aminoethyl]-benzenesulfonyl fluoride). The lysis buffer plus 10 mM sodium fluoride and 1% phosphatase inhibitor cocktail (Sigma-Aldrich, St Louis, MO) was used to prepare the protein extraction for phosphoprotein assay. The protein concentration of the lysates was estimated using the method of BCA (with reagents from Pierce; Rockford, IL), and the total protein content between samples was equalized. Whole cell protein extract lysates were used. The protein was separated by SDS-PAGE on 8% gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 10% fat free milk or 5% BSA and then incubated overnight at 4°C or 2 hours at 23°C with the primary antibodies (anti-Nav1.8 1:500, anti-Nav1.7 1:1000, and anti-GAPDH 1:5000, both from Sigma Co., St Louis, MO; anti-COX-2 1:1000 from Cayman Chemical, Ann Arbor, MI; anti-NF-κB/p65 1:1000 and anti-phospho-NF-κB/p65 1:1000 from Cellsignalling Tech Inc., Danvers, MA). The blots were developed using a SuperSignal West Femto Kit (Pierce) with horseradish peroxidase-conjugated secondary antibodies (R&D System, Minneapolis, MN). Data were analyzed with the Molecular Imager (ChemiDoc XRS; Bio-Rad Laboratories) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories).

IL-1β Determination 

To detect IL-1β in DRG tissues, the DRG homogenate was assayed using an ELISA-based kit (Rat IL-1β/IL-1F2 Quantikine ELISA kit; R&D System) according to the manufacture's protocol. Proteins were quantified (BCA protein Assay; Pierce) using the samples for Western blot analysis as described in the last paragraph. All IL-1β determinations were performed in duplicate serial dilutions.

Hematoxylin and Eosin (HE) Staining of Inflamed Ganglion 

The L5 DRGs were taken from rats at different periods of time after surgery. Appearance of the ganglia was observed under light microscope (×4) and higher magnification (×40) before electrophysiological recordings (14–15 postoperative days). From another 15 rats (5 in each of the groups: control, IVFI+CIC, and IVFI+Vehicle), the bilateral L5 DRGs were removed on the 21 postoperative days after the rats were anesthetized and perfused with 100 mL of heparinized saline followed by 400 mL 4% paraformaldehyde in phosphate buffer. The ganglia were postfixed in the same fixative for 3 hours, and then immersed in 30% sucrose overnight at 4°C. Frozen tissues were sectioned (thick 15 μm; Leica CM1850-3-1; Leica Microsystem, Germany), stained with HE. We chose 4 (2nd, 4th, 6th, and 8th) out of the total 10 sections from the layer of cells (∼200 μm) in each ganglion to do further microscope analysis of the glial cells. Four grid areas were chosen from within each section. Each grid area (4 cm2 under ×10 microscope) was used to count the glial cells those located on the surface of the sections and could be identified under higher magnification (×40). The counts were then calculated and converted into the numbers of glial cells in units of 100 × 100 μm2 and expressed in Fig 4G in the Results.

Topical Application of CIC 

CIC and its vehicle cream were obtained from Core Products Intl., Inc (CPII, Osceola, WI; it is unavailable on the market). The cream (each ∼.54 g) was applied onto the shaved area of the surface of back skin (∼2 × 4 cm2), which covered the inflamed L5 IVF and the adjacent area. A series of 5 applications were initiated 3 days after surgery, and subsequently applied daily for 5 consecutive days.

Statistical Analysis 

Differences in difference scores of the latency and mechanical threshold over time were tested with 2-way repeated measure analyses of variance (ANOVA) followed by post hoc paired comparisons. One-way ANOVA followed by Dunnett's tests were used to test the hypothesis that resting membrane potential and excitability of DRG neurons as well as the Western blot analysis of expression of Nav1.7 and Nav1.8, COX-2 and NF-κB protein, and/or phosphorylation, and IL-1β level in CIC-treatment groups were significantly different from the IVFI groups, but not the sham. Individual t-tests were used to test specific hypotheses about differences between each IVFI or with CIC treatment group and its corresponding control group for each electrophysiological parameter tested. All data are presented as mean ± SEM. Unless otherwise stated, statistical results described as significant are based on a criterion of a P value of less than .05.

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Results 

Repetitive, Topical Application of CIC Suppresses IVFI-Induced Thermal Hyperalgesia and Mechanical Allodynia 

The animals that received L5 IVF injection of IS exhibited thermal hyperalgesia and mechanical allodynia. As shown in Fig 1, the foot ipsilateral to the IVFI became more sensitive to the thermal or mechanical stimulus, but the responses of the foot contralateral to IVFI were not significantly changed (data not shown). Repetitive, topical application of CIC significantly reduced the severity and duration of IVFI-induced thermal hyperalgesia (Fig 1A) and mechanical allodynia (Fig 1B). Severity of thermal hyperalgesia started to decrease significantly after 3 to 4 applications, evidenced by the increased latency of foot withdrawal to heat stimulation. The thermal hyperalgesia was completely inhibited following 4 to 5 doses of CIC treatment. Such inhibition lasted to the last test on the 35th postoperative day (Fig 1A). The mechanical allodynia was significantly reduced by approximately 50% (Fig 1B) after 3 to 4 doses of CIC treatment. The vehicle treatment did not alter the thermal and mechanical sensitivity in the feet ipsilateral and contralateral to IVFI and in sham animals.

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  • Figure 1 

    Effects of repetitive, topical application of a compound ibuprofen cream, CIC, on thermal hyperalgesia and mechanical allodynia following intervertebral foramen inflammation (IVFI). (A) Effects of CIC treatment on IVFI-induced thermal hyperalgesia. (B) Effects of CIC treatment on IVFI-induced mechanical allodynia. Number of rats used in each group in A and B: Sham = 8, IVFI = 8, IVFI + Vehicle = 10, IVFI + CIC = 10. Data are expressed as Mean ± S.E.M. ∗P < .05, ∗∗P < .01 compared to the group of sham; #P < .05, ##P < .01 compared to groups of IVFI and IVFI + Vehicle.

Repetitive, Topical Application of CIC Inhibits IVFI-Induced DRG Neuron Hyperexcitability 

Behavioral hyperalgesia and allodynia can be immediately modulated by excitability of the sensory neurons located within the DRG. Our electrophysiological studies showed that the 3 categories of the DRG neurons, the large- and medium-sized and small neurons, in the inflamed DRG were hyperexcitable. Such hyperexcitability was significantly inhibited by CIC treatment. Examples of electrophysiological responses to intracellular test stimuli applied to DRG neurons and the CIC treatment effects are shown in Figs 2A–C and data summarized in Figs 2D–F. In the inflamed DRG neurons, the mean AP current threshold decreased significantly in neurons of all sizes (P < .01, Fig 2D). Hyperexcitability of DRG neurons was also revealed as an enhancement of repetitive discharge evoked by a 50-ms depolarizing current pulse normalized to AP current threshold. Fifty to 70% of the neurons from inflamed DRG responded with 3 or more APs to the normalized depolarizing current, and the rest exhibited 1 or 2 APs. In contrast, only about 20% of the neurons from control DRG discharged 3 or more APs (P < .01 in each case; Fig 2E). The average neural discharges increased significantly in the inflamed DRG (Fig 2F). In addition, the inflamed DRG neurons were significantly depolarized (Fig 2G). A week after CIC treatment (14–15 postoperative days), the IVFI-induced hyperexcitability was significantly reduced and depolarization of the membrane potential largely recovered (Figs 2A–G).

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  • Figure 2 

    Effects of repetitive, topical application of CIC on resting membrane potential (RMP) and excitability of DRG neurons. (A-C) Examples of action potentials (AP) and cellular responses in DRG neurons evoked by the intracellular test protocol; left panel, hyperpolarizing and depolarizing responses to 50-ms current pulses (the largest depolarizing pulse reached threshold for evoking an action potential; only last 2 pulses and cellular responses are shown); right panel, discharge patterns in DRG neurons tested with 1-second pulses delivering 2.5 × threshold current as described in Methods. (D-G) CIC treatment reversed the decreased AP current threshold (D), increased incidence of repetitive discharge (E and F) and membrane depolarization (G) in large- and medium-sized and small DRG neurons following IVFI. DRG neuron excitability was tested 1 week after CIC treatment, ie, 2 weeks after IVFI. Number of cells in each group: large-sized cells = 26; 23 and 25 in Sham, IVFI + Vehicle and IVFI + CIC, respectively; medium-sized cells = 28; 26 and 26 in Sham, IVFI + Vehicle and IVFI + CIC, respectively; small cells = 17, 20 and 20 in Sham, IVFI + Vehicle and IVFI + CIC, respectively. ∗∗P < .01 compared to sham; #P < .05, ##P < .01 compared to group of IVFI + Vehicle.

Repetitive, Topical Application of CIC Suppresses IVFI-Induced Upregulation of Expression of Nav1.7 and Nav1.8 

Nerve injury and inflammation can cause alteration of VGSCs, which are necessary for electrogenesis and nerve impulse conduction. The VGSCs, Nav1.7 and Nav1.8 are considered to be tremendously important in modulation of the DRG neuron excitability and behavioral hyperalgesia.6, 17, 32, 46 Using Western blot analysis, we examined expression of the Nav1.7 and Nav1.8 protein in the inflamed DRG with or without CIC treatment. The results showed that expression of Nav1.7 and Nav1.8 protein in the inflamed DRG were significantly increased, and upregulation of these molecules was significantly reduced following the repetitive CIC treatment. Examples are given in Fig 3A and data summarized in Fig 3B.

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  • Figure 3 

    Western blot analysis of expression of Nav1.7 and Nav1.8 protein in inflamed ganglia with treatment of CIC or CIC vehicle. (A) Representative Western blot illustrating differential expression of Nav1.7 and Nav1.8 protein following different treatment. (B) Data summary of expression of Nav1.7 and Nav1.8 protein. Changes are standardized by protein level in the corresponding control group. Three samples from 3 rats included in each group. Tissues were collected 1 week after CIC treatment, ie, 2 weeks after IVFI. ∗∗P < .01 compared to the corresponding sham group; #P < .05 compared to the corresponding group of IVFI.

Repetitive, Topical Application of CIC Improves Pathological Signs of the Inflamed Ganglion 

Under the light-dissecting microscope, the ganglion from the inflamed IVF showed clear signs of inflammation. As shown in Figs 4 A–C, the DRG appeared to be covered by a layer of connective tissue that was somewhat difficult to remove, and the increased vascularization could be seen on the surface of the ganglia. In contrast, the ganglion contralateral to IVFI or from naive or sham-operated animals looked clear without obvious blood vessels. HE staining further showed the inflammatory signs in the inflamed DRG. Satellitosis, which was not found in the control ganglion slices (Fig 4D), was present in most of the slices from inflamed ganglia (an example given in Fig 4E). Satellitosis is a condition marked by an accumulation of glial cells around the neurons and is often a prelude of the neuronophagia (phagocytosis of nerve cells). Neuronophagia and karyopyknosis (cytologic characteristics of the superficial or cornfield cells of stratified squamous epithelium in which there is shrinkage of the nuclei and condensation of the chromatin into structureless masses) were sometimes observed in the inflamed, but not the control ganglia. These signs of the DRG inflammation were significantly reduced a week after termination of the repetitive, topical application of CIC (Fig 4F). Alteration of the glial cells that surrounded the DRG neurons following IVFI with CIC treatment or the vehicle is summarized in Fig 4G.

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  • Figure 4 

    Appearance and pathological manifestations of DRGs and the neurons within. (A-C) Images under light dissecting microscope. (A) The L5 ganglion contralateral to the inflamed DRG. (B) The inflamed L5 ganglion with increased vascularization on the surface. (C) The inflamed L5 ganglion after IVFI with CIC treatment. (D-F) HE stained DRG cells. (D) L5 ganglion contralateral to the inflamed ganglion. The DRG neurons (bigger cells sized in diameter from ∼10–60 μm) and the glial cells are shown. (E) Inflamed L5 ganglion. A large amount of glial cells surrounded the DRG neurons and formed the phenomenon of “Satellitosis” (arrows). (F) The inflamed L5 ganglia 3 weeks after CIC treatment. Scales: 1 mm (left) and 30 μm (right). (G) Summary of effects of CIC treatment on the inflamed DRG neurons characterized with changes in the amount of glial cells. DRGs were examined 2 weeks after CIC treatment, ie, 3 weeks after IVFI. ∗P < .01 compared to group of sham; #P < .05 compared to group of IVFI + Vehicle.

Repetitive, Topical Application of CIC Suppresses IVFI-Induced Increases in Expression of NF-κB and COX-2 and Level of IL-1β in Inflamed Ganglia 

Given that CIC treatment can reduce signs of inflammation of the ganglion, we further examined effects of CIC treatment on phosphorylation and expression of the transcription factor NF-κB and expression of the pro-inflammatory enzyme COX-2 using Western blot analysis, and induction of cytokine IL-1β determined by ELISA. These molecules are considered important in inflammation and chronic pain.1, 28 Expression of NF-κB/p65 protein and its level of phosphorylation (p-NF-κB/p65) (Figs 5A, B), expression of COX-2 protein (Figs 5C, D) shown by Western blot analysis, and level of IL-1β measured by ELISA (Fig 5E) significantly increased in the inflamed DRG. Repetitive, topical treatment of CIC, but not the vehicle control, significantly reduced the increased activity of NF-κB, expression of COX-2, and level of IL-1β (Fig 5).

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  • Figure 5 

    Effects of IVFI and CIC treatment on expression of NF-κB and COX-2 and induction of IL-1β in inflamed ganglia. (A) Representative Western blot illustrating alteration of expression of NF-κB/65 protein and its level of phosphorylation (p-NF-κB/65) following different treatments. (B) Data summary of NF-κB/65 and p-NF-κB/65. (C) Representative Western blot illustrating alteration of expression of COX-2 protein following different treatments. (D) Data summary of COX-2. (E) Changes of IL-1β in DRGs, assayed using ELISA-based kits, following different treatments. Three samples from 3 rats were included in each group in A-E. ∗P < .05, ∗∗P < .01 compared to the corresponding sham group; #P < .05 compared to the corresponding group of IVFI.

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Discussion 

The present study provides the first evidence that an anti-inflammatory reagent, CIC, can be used topically to suppress pain due to IVFI and/or DRG inflammation through inhibition of sensory neuron hyperexcitability and the immune and inflammatory responses. CIC treatment significantly reduces severity and shortens duration of thermal hyperalgesia and mechanical allodynia, inhibits DRG neuron hyperexcitability and the associated upregulation of expression of Nav1.7 and Nav1.8 protein, and reduces manifestations of DRG inflammation and the associated upregulation of expression of transcription factor NF-κB and proinflammatory enzyme COX-2 and level of IL-1β in the affected DRGs. This study suggests a convenient and safe clinical intervention for treating pain due to intervertebral foramen inflammation and the similar syndromes.

Inflammatory response contributes to the peripheral and spinal central sensitization and plays a key role in the pain manifestations in inflammatory pain as well as in neuropathic pain.1, 5, 39, 40, 42, 43, 44 Following peripheral inflammation or tissue/nerve injury, chemical factors such as cytokines, nerve growth factors, inflammatory mediators, and other substances release and activate or change the properties of DRG neurons and spinal dorsal horn neurons as well as increase their excitability and, therefore, contribute to pain and/or hyperalgesia.1, 5, 39, 40, 42, 43, 44 In the present study, the DRG neuron inflammation was produced by injection of IS into the IVF that can produce an immediate effect on the DRG neurons and also to the constituents within the IVF, ie, nerve root, blood, and lymph vessels. This IVFI-induced inflammatory response may be somewhat different from the secondary inflammatory responses of DRG neurons due to the distal tissue/nerve inflammation/injury. Studies have shown different alterations of expression of COX-2, K+ current, and both TTX-S and TTX-R Na+ currents in the affected DRG neurons following IVF local inflammatory stimulus and the distal peripheral inflammation and nerve lesion.2, 17, 52 Peripheral sensitization is important for development of inflammatory pain.35, 55 Treatment effects of anti-inflammatory reagents on the peripheral sensitization have been investigated in models of the distal tissue inflammation and sciatic nerve lesion. Here we demonstrate, for the first time, that topical application of an anti-inflammation reagent on the back skin covering the inflamed IVF area can effectively alleviate thermal hyperalgesia and mechanical allodynia due to IVFI and/or DRG inflammation. These results suggest that CIC and probably many other analgesic reagents that are similar to CIC could be used topically, the most safe and convenient intervention, to relieve pain syndromes in similar clinical disorders.

The increased excitability of DRG neurons have been demonstrated to contribute greatly to behavioral hyperalgesia. Our electrophysiological studies demonstrate that CIC treatment significantly inhibits IVFI-induced DRG neuron hyperexcitability by reversing the decreased AP current threshold, depolarized membrane potential, and increased spontaneous and evoked neural discharges. The VGSCs are necessary for electrogenesis and nerve impulse conduction and considered to be important in modulation of DRG neuron excitability and behavioral hyperalgesia.6, 17, 18, 32, 44, 48 VGSCs can be dynamically regulated after nerve injury, DRG compression, or peripheral inflammation and play important roles in modulating neural excitability.6, 17, 32, 48 In DRG neurons, these VGSCs include Nav1.8 and Nav1.9, which conduct tetrodotoxin-resistant currents, and Nav1.3, Nav1.6, and Nav1.7, which conduct tetrodotoxin-sensitive currents. The VGSCs, Nav1.7 and Nav1.8 are considered the most important and a target of some analgesics. Studies have shown that interfering with expression of Nav1.7 and/or Nav1.8 can effectively suppress neuropathic pain.6, 8, 18, 21 Here we show that CIC treatment significantly inhibits IVFI-induced upregulation of Nav1.7 and Nav1.8. These results suggest that suppression of the sodium channels Nav1.7 and Nav1.8 may contribute to inhibition of DRG neuron hyperexcitability and the analgesic effect following CIC treatment in IVFI rats. This result is supported by a study that ibuprofen can modulate Nav1.7 and Nav1.8 channels in a peripheral inflammation pain model.12

In addition to alleviating inflammatory hyperalgesia, inhibiting DRG neuron hyperexcitability and suppressing sodium channel activity, CIC treatment significantly improves pathological signs of the DRG inflammation. The faster elimination of DRG inflammation may contribute greatly to recovery of the DRG neuron excitability and the thermal and mechanical sensitivity. Our results further show that CIC treatment can modulate activity of NF-κB protein and therefore reduce the DRG inflammation. The transcription factor NF-κB is composed of homo- and hetero-dimers of different Rel family proteins (p65, RelB, c-Rel, p52, and p50) and activated by a huge array of stimuli including inflammatory cytokines recognized by specific membrane receptors such as tumor necrosis factor receptor.11, 13 NF-κB is a crucial regulator of many physiological and pathophysiological processes, including control of the adaptive and innate immune responses, inflammation, proliferation, tumorigenesis, and apoptosis.28 Thus, the tight regulation of NF-κB activity within a cell is critically important. A number of recent studies indicate that activation of NF-κB is involved in the pathogenesis of neuropathic pain and inflammatory hyperalgesia. Phosphorylation of the p65 subunit of NF-κB was required in these pathways.28, 29 Further, expression of the proinflammatory enzyme COX-2 and induction of the cytokine IL-1β in the inflamed DRG tissues are also suppressed by the CIC treatment. COX-2 is a key enzyme to produce prostaglandins. Both COX-2 and IL-1β can be regulated by NF-κB and involved in the processing of inflammation.16, 28 The observed alteration of COX-2 and IL-1β in DRG may be induced directly by IVFI and CIC treatment or as a subsequent change following alteration of the transcription factor NF-κB. These findings suggest that inhibition of NF-κB, COX-2, and/or IL-1β contributes to CIC treatment-induced elimination of the DRG inflammation.

CIC used here consists of ibuprofen, glucosamine, chondroitin, methylsulfonylmethane, and bromelain, and is clinically formulated with the proprietary liposomal dermal delivery matrix. Of these components, ibuprofen, one of the nonsteroidal anti-inflammatory drugs (NSAIDs), is actually a nonselective COXs including COX-2 inhibitor. Glucosamine and chondroitin are used in combination for osteoarthritis pain treatment due to stimulation of the synthesis of cartilage glycosaminoglycans and proteoglycans. It has been reported that glucosamine can inhibit the inflammatory response by its inhibitory effects on free radical scavenging, pro-inflammation mediators' expression, such as IL-8, matrix-metalloproteinase (MMP)-2 (MMP-2) and MMP-9, intercellular adhesion molecule-1, and tumor necrosis factors-α.16, 25, 31, 51, 56 Bromelain treatment can attenuate inflammation by decreasing secretion of pro-inflammatory mediators and neutrophil migration to sites of inflammation, and expression of COX-2.3, 9, 30, 36 Methylsulfonylmethane exhibits anti-inflammatory effects on lipopolysaccharide-induced inflammatory responses.20 Therefore, CIC treatment may inhibit multiple aspects of inflammatory responses in addition to inhibiting the sensory neuron excitability, the sodium channel expression and activity of NF-κB, expression of COX-2, and the cytokine IL-1β in the inflamed ganglion.

Several lines of studies have compared topical ibuprofen gel and oral ibuprofen in clinical experimental pain paradigms and concluded that topical application of NSAIDs produces analgesia in models of cutaneous pain24, 33, 34 and muscle pain.33, 47 In a clinical context, several reviews have all concluded that there was clear evidence to support efficacy of topical NSAIDs given by gel, spray, or patch for conditions of soft-tissue injuries (eg, sprains, strains, tendonitis) and rheumatic diseases,14, 26, 33, 50, 54 and for sports-related soft-tissue injury.10 Our study is the first to demonstrate that CIC, a cream similar to the NSAIDs gel, applied to the skin surface above the affected IVF can effectively relieve the pain due to IVFI. Studies have further shown that the bioavailability and plasma concentrations following topical application are 5 to 15% of those achieved by systematic delivery.15 When NSAIDs are administrated topically, relatively high concentrations occur in the dermis, whereas levels in the muscle are at least equivalent to those following systematic administration.15 Topical applied NSAIDs do reach the synovial fluid, but it is not clear whether this reflects local penetration or results from systematic circulation.50 Another advantage is that 2 peaks of blood concentration of ibuprofen are found in 2 to 3 hours and 7 to 10 hours after topical application of NSAIDs. In contrast, only 1 high peak concentration appears after oral administration.49 Therefore, we believe that, in the present study, the CIC applied on the skin surface covering the inflamed IVF and DRG may exert its analgesic effect via similar pathways, local penetration, and systematic circulation although we were not able to measure blood concentrations of CIC components such as ibuprofen. Detailed pharmacokinetic studies are required to answer this question.

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Conclusion 

The present study demonstrates that repetitive, topical application of a compound ibuprofen cream, CIC, can effectively suppress severity and shorten duration of thermal hyperalgesia and mechanical allodynia caused by lumbar IVFI with DRG neuron inflammation. This treatment effect may result from CIC-induced faster elimination of the DRG neuron inflammation and recovery of DRG neuron excitability. Inhibition of the inflammation-induced upregulation of sodium channels Nav1.7 and Nav1.8 may contribute to recovery of the DRG neuron excitability, while inhibition of activity of NF-κB, expression of COX-2 and induction of IL-1β may contribute to suppressing the immune and inflammatory responses. This study suggests a convenient and safe intervention for clinical treatment of pain due to IVFI and/or the similar syndromes.

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Acknowledgments 

We thank P. Mattison for stimulating discussion, R. Natividad Jr., J. Feiro, and M. Dominguez for their assistance in the experiment and lab management. The compound ibuprofen cream (CIC) and the vehicle cream were provided by Core Products International Incorporation (CPII).

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 Supported by Parker Research Foundation (PCCRF-BSR0905), CPII (CPII-XJS0809), and National Institute of Health (NIH-1R43AT004933-01).

PII: S1526-5900(10)00582-1

doi:10.1016/j.jpain.2010.06.008

The Journal of Pain
Volume 12, Issue 1 , Pages 141-152, January 2011

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