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Knockdown of Synaptic Scaffolding Protein Homer 1b/c Attenuates Secondary Hyperalgesia Induced by Complete Freund's Adjuvant in Rats

Yao, Yong-Xing MD*; Jiang, Zhen MD; Zhao, Zhi-Qi PhD

doi: 10.1213/ANE.0b013e31822c0b98
Analgesia: Research Reports
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BACKGROUND: Previous studies have demonstrated that Homer 1b/c, a postsynaptic molecular scaffolding protein that binds and clusters metabotropic glutamate receptors at neuronal synapses, has an important role in the metabotropic glutamate receptor signaling process. In the current study, we investigated the possible involvement of Homer 1b/c in secondary hyperalgesia induced by complete Freund's adjuvant (CFA).

METHODS: Chronic inflammation was induced by injecting CFA into the left hind ankle of Wistar rats. Homer 1b/c antisense or missense oligonucleotides were intrathecally administrated (antisense, 10 μg/10 μL, 5 μg/10 μL, or 2.5 μg/10 μL, once a day; missense, 10 μg/10 μL) from 5 to 8 days after the onset of inflammation. The withdrawal threshold and withdrawal latency to mechanical or thermal stimuli were determined before and after the intrathecal administration. The expression and distribution of Homer 1b/c were examined in the spinal cord using immunological techniques.

RESULTS: Mechanical allodynia and thermal hyperalgesia were induced within 24 hours and maintained for >2 weeks after the CFA injection. The expression of Homer 1b/c reached the highest level 7 days after inflammation and returned to baseline at day 28. Intrathecal administration of Homer 1b/c antisense oligonucleotides markedly reduced the expression of Homer 1b/c protein in the spinal cord. Additionally, administration of Homer 1b/c antisense oligonucleotides attenuated secondary mechanical hypersensitization on days 2 to 5 and reduced thermal hypersensitization on days 3 to 4. There were no effects of missense oligonucleotides on hypersensitization and the expression of Homer 1b/c. In the naïve rats, Homer 1b/c antisense oligonucleotides did not affect the mechanical and thermal responses or locomotor activity.

CONCLUSIONS: These novel results demonstrate that Homer 1b/c in the spinal cord contributes to the maintenance of secondary hyperalgesia induced by CFA and suggest that Homer 1b/c may be a novel target for pain therapy.

Published ahead of print October 14, 2011 Supplemental Digital Content is available in the text.

From the *Department of Anesthesiology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou; Department of Anesthesiology, Institute of Shanghai Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai; and Unit of Pain Research, Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China.

Supported by the National Basic Research Program of China grant 2006CB500807, and National Natural Science Foundation of China grants 30830044 and 30972846.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Zhi-Qi Zhao, PhD, Unit of Pain Research, Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Rd., Shanghai 200032, China. Address e-mail to zqzhao@fudan.edu.cn.

Accepted June 16, 2011

Published ahead of print October 14, 2011

Homer 1b/c, a molecular scaffolding protein in the postsynaptic density (PSD) of excitatory synapses, has been shown to attach metabotropic glutamate receptors (mGluRs) to internal signaling molecules, including intracellular calcium stores.1,2 This suggests that Homer 1b/c may be involved in many physiological and pathophysiological functions triggered via the activation of mGluRs in the central nervous system. Repeated cocaine administration has been demonstrated to attenuate group I mGluR-mediated glutamate release and behavioral activation, which is associated with a selective reduction in Homer 1b/c protein in the nucleus accumbens.3 The behavioral sensitization to cocaine arises in part because of a reduction in Homer 1 gene products in the accumbens.4 More importantly, spinal Homer 1a and Homer 1b/c were associated with neuropathic pain induced by chronic compression of the dorsal root ganglion and chronic constriction injury of the sciatic nerve in rats.57 However, it is unclear whether Homer 1b/c contributes to the maintenance of secondary hyperalgesia from inflammatory pain. In the present study, we investigated the changes in Homer 1b/c expression after monoarthritis inflammation and the effects of Homer 1b/c protein deficiency in the spinal cord after pain sensitization induced by complete Freund's adjuvant (CFA).

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METHODS

Animal Housing and Preparation

Male Wistar rats (weighing approximately 200 g on arrival) were obtained from the Chinese Academy of Sciences animal center and housed 2 per cage with water and food available ad libitum. A 12-hour light/12-hour dark cycle was used, with lights on at 08:00. All animal experiments were performed with the approval of the Animal Care Committee at Fudan University and were consistent with the ethical guidelines for investigating experimental pain in animals.8 All efforts were made to minimize the number of animals used and their suffering. Animals were acclimated to the housing facility for 3 days before starting the experiments.

The rats were implanted with an intrathecal catheter under intraperitoneal anesthesia (chloral hydrate, 200 mg/kg). A polyethylene-10 catheter (Becton Dickinson, Sparks, MD) was inserted into the subarachnoid space at the level of the spinal cord lumbar enlargement according to the method described by Størkson et al.9 The animals were allowed to recover for 3 days before being used experimentally. Rats showing postoperative neurological deficits were excluded from the study. The animals were randomly divided into groups.

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Induction of Chronic Inflammation

Arthritis was induced by injecting 50 μL CFA (Santa Cruz Biotechnology, Santa Cruz, CA) into the left tibiotarsal joint, under brief isoflurane anesthesia, as described by Butler et al.10 The control animals were injected with 50 μL saline. During the course of arthritis, the inflammatory reaction was followed and scored according to the method of Castro-Lopes et al.11

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Intrathecal Homer 1b/c Antisense Oligonucleotide Administration

Oligonucleotide sequences (Sangon, Shanghai, China) were dissolved in sterile saline, and the concentration was determined by optical density measurement. Both the antisense (AS) and missense (MS) oligonucleotide sequences were 25 bases in length and were 5′ and 3′ tailed with 5 phosphorothioated nucleic acids to protect them from degradation by nucleases. The sequences of the Homer 1b/c AS and MS oligonucleotides were from Ghasemzadeh et al.4: AS 5′-GGAAGACATGAGCTCGAGTGCTGAA-3′; MS 5′-TGACTAGGTCTCGTTACCTGTCCTC-3′. The rats were injected intrathecally with saline (10 μL), AS oligonucleotides (2.5 μg, 5 μg, or 10 μg/10 μL), or MS oligonucleotides (10 μg/10 μL), followed by an injection of 10 μL saline to flush the catheter, every 24 hours for 4 days.

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Behavioral Testing

Before and after the intrathecal oligonucleotide injection, all animals were behaviorally tested to determine the hindpaw withdrawal threshold to mechanical stimuli. In brief, the animals were placed in a cage with a wire mesh floor and allowed to explore and groom until they settled. A set of von Frey hairs (Stoelting Co., Wood Dale, IL) with bending forces ranging from 1.0 to 15 g was applied to the plantar of the inflamed hindpaw in ascending order. Brisk withdrawal or paw flinching was considered a positive response. The bending force of the von Frey hair triggering the withdrawal of the hindpaw was recorded. Three measurements were made from each animal, and the average value of the 3 measurements was calculated.12

The hindpaw withdrawal latency to noxious heat stimuli was determined with a Tail Flick Analgesia Meter (Model 33B; IITC Life Science Inc., Woodland Hills, CA), as described by Hargreaves et al.13 Briefly, the rats were placed in a Plexiglas chamber on a glass plate under which a light box was located. A radiant heat stimulus was applied by aiming a beam of light through a hole in the light box onto the heel of each hindpaw through the glass plate. The light beam was turned off when the rat lifted its foot, and the time between when the light beam was turned on and the foot was lifted was measured. This time was defined as the hindpaw withdrawal latency. Each trial was repeated 3 times at 5-minute intervals. A cutoff time of 20 seconds was used to avoid tissue damage.14

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Locomotor Function

The effects of Homer 1b/c AS oligonucleotides on locomotor functions were examined on the fifth day after pretreatment with the AS oligonucleotides. The following tests were performed according to the method of Coderre and Van Empel15: Scores for placing, grasping, and righting reflexes were based on counts of each normal reflex exhibited in 6 trials. In addition, the rat's general behaviors, including spontaneous activity, were observed.

  1. Placing reflex: The rat was held with its hindlimbs slightly lower than its forelimbs, and the dorsal surfaces of the hindpaws were brought into contact with the edge of a table. The experimenter recorded whether the rat placed its hindpaws on the table surface reflexively.
  2. Grasping reflex: The rat was placed on a wire grid and the experimenter recorded whether the hindpaws grasped the wire on contact.
  3. Righting reflex: The rat was placed on its back on a flat surface and the experimenter noted whether it immediately assumed the normal upright position.
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Tissue Preparation and Immunohistochemistry

Seven days after inflammation, the rats were given an overdose of chloral hydrate (400 mg/kg, intraperitoneally). Animals were perfused through the ascending aorta with 250 mL saline (37°C) followed by 750 mL of 4% paraformaldehyde (4°C). The spinal cords were harvested and postfixed in the same fixative solution for 4 hours and then cryoprotected overnight (30% sucrose in 0.1 M phosphate buffer). The caudal portion of the spinal cord segments L4 and L5 was dissected, and 40-μm, free-floating transverse sections were cut with a freezing microtome. The sections were then stored at −20°C in a cryoprotective solution until further processing.

For immunocytochemistry, the sections were thawed, washed in phosphate-buffered saline (PBS), and then incubated for 2 hours in 10% normal donkey serum. Afterward, the sections were incubated with rabbit Homer 1b/c antibody (Santa Cruz Biotechnology) at a concentration of 1:200 for 48 hours at 4°C. The sections were then washed 3 times with a solution of PBS and subsequently incubated for 2 hours in antirabbit antiserum (1:200). The sections were then thoroughly washed with PBS, mounted on gelatin-coated slides, cleared in xylene, and coverslipped with Eukitt. Control sections lacking primary antibody were stained in parallel. No labeling was observed in the control sections.

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Protein Extraction

Rats were decapitated immediately under deep anesthesia. The spinal lumbar enlargements were quickly excised and divided into ipsilateral and contralateral halves. The ipsilateral half was further divided into dorsal and ventral quadrants. The ipsilateral dorsal quadrants were homogenized in ice-cold homogenization buffer (1:10, w/v) consisting of 50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), 100 mM KCl, 320 mM MgCl2, 0.2 mM dithiothreitol, phosphatase, and protease inhibitors (20 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 50 mM NaF, 0.5 mM EDTA, 1 mM each of EGTA, phenylmethylsulfonyl fluoride, and benzamidine, and 5 μg/mL each of aprotinin, leupeptin, and pepstatin A). The homogenates were centrifuged at 2500g for 10 minutes at 4°C. The supernatant was collected, and the protein concentrations were determined with a Micro BCA™ Protein Assay reagent kit (Pierce, Rockford, IL).

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Immunoprecipitation

Samples were preincubated for 1 hour with 20 μL protein G agarose, and then centrifuged to remove any protein that adhered nonspecifically to the protein G. The supernatants were incubated with 1 μg rabbit polyclonal Homer 1b/c antibody overnight at 4°C. Thirty μL protein A was then added to the reaction system, and the incubation continued for 3 hours. Samples were centrifuged at 800g, and the pellets were washed 3 times with CHAPES buffer. Bound proteins were eluted by adding 1× sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer (10 μL). The proteins were then boiled at 100°C for 5 minutes, and centrifuged at 5000 rpm for 10 seconds. The supernatants were collected for immunoblotting.

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Immunoblot Analysis

Proteins in the sample were separated using a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, and then transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) using a wet transfer apparatus (Bio-Rad Mini, Hercules, CA). Proteins bound to the membrane were stained with Ponceau S solution (Sigma, St. Louis, MO) to determine the transfer quality. Membranes were blocked for 2 hours at room temperature and incubated with the primary antibody to postsynaptic density protein-95 (PSD-95) (1:200; Santa Cruz Biotechnology), Homer 1b/c (1:2000; Chemicon, Temecula, CA), or β-tubulin (1:8000; Sigma-Aldrich, St. Louis, MO) in antibody buffer containing Tween-20 (50 μL/100 mL) overnight at 4°C. The membranes were washed with Tris-buffered saline with Tween-20 and incubated with peroxidase-conjugated secondary antibody for 2 hours at room temperature. The membranes were washed thoroughly, and the immunolabeling was visualized with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and exposure to Kodak film. The intensity of the blots was quantified with densitometry.

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Statistical Analysis

Data are expressed as the mean ± SEM and were statistically analyzed with 1-way or 2-way analysis of variance followed by Student-Newman-Keuls or Dunnett multiple comparison test. The effect of AS or missense treatment on protein levels was compared using independent t test. A P value <0.05 was considered to be statistically significant.

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RESULTS

In agreement with our previous study,16 rats developed stable monoarthritis in the left ankle after injection of CFA. Generally, swollen ankles and immobility of the inflamed legs were observed. The inflammatory score reached its maximum value4 within 24 hours and was maintained for >7 days. After this period, the inflammatory score showed a slight reduction (data not shown). Parallel to the development of inflammation, there was significant secondary mechanical allodynia and thermal hyperalgesia (Fig. 1). In contrast with CFA, saline injection failed to induce inflammatory signs and secondary hyperalgesia.

Figure 1

Figure 1

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Abundant Expression of Homer 1b/c in the Superficial Dorsal Horn of the Spinal Cord

The expression and distribution of Homer 1b/c were examined in the spinal cord with immunological techniques. Using immunohistochemistry, we found that Homer 1b/c immunoreactivity had a higher density in laminae I and II and outer lamina III than in other laminae of the spinal dorsal horn (Fig. 2, A and B). Under high magnification, Homer 1b/c immunoreactivity is located in the cell bodies, fibers, and some terminals. The positive cells are small (<20 μm) and appear oval, fusiform, or round with some processes (Fig. 2C, arrows). Most of the cells are distributed in laminae I to III. Immunoblotting analysis confirmed the enriched expression of Homer 1b/c in the spinal cord. Abundant Homer 1b/c protein was detected in the dorsal horn, but nearly no Homer 1b/c protein was detected in the ventral horn (Fig. 2D).

Figure 2

Figure 2

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Monoarthritis-Induced Upregulation of Homer 1b/c Expression in the Ipsilateral Dorsal Horn

Next, we investigated whether Homer 1b/c expression in the dorsal horn changed after monoarthritis induced by CFA injection. Unilateral, intraarticular injection of CFA induced a significant increase in the expression of Homer 1b/c in the ipsilateral spinal dorsal horn (P < 0.05), where Homer 1b/c expression reached the highest level at day 7, and returned to baseline at day 28. The expression of Homer 1b/c did not significantly change in the contralateral dorsal horn (Fig. 3).

Figure 3

Figure 3

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Homer 1b/c AS Oligonucleotides Reduced Homer 1b/c Protein Expression and Attenuated Pain Hypersensitization

To address the role of Homer 1b/c protein in the maintenance of inflammatory pain, we examined Homer 1b/c deficiency in the spinal cord by using Homer 1b/c AS oligonucleotides. Homer 1b/c AS oligonucleotides (10 μg/10 μL), missense oligonucleotides (10 μg/10 μL), or saline (10 μL) was intrathecally injected every 24 hours for 4 days from day 5 to day 8 postinflammation. The lumbar enlargement segments of the spinal cord were harvested on day 9 postinflammation. Western blot analysis showed that the AS oligonucleotides markedly reduced the expression of Homer 1b/c in the spinal lumbar enlargement segments, but the missense oligonucleotides produced no effect (Fig. 4A). Immunohistochemical analysis confirmed the reduction of Homer 1b/c and revealed that the AS oligonucleotides failed to alter the expression of NeuN and glial fibrillary acidic protein, 2 markers for neurons and astrocytes, respectively (Fig. 4B).

Figure 4

Figure 4

To evaluate the role of Homer 1b/c protein in the secondary mechanical and thermal hyperalgesia observed after monoarthritis, the same intrathecal injections were made every 24 hours for 4 days from day 5 to day 8 postinflammation. The results showed that Homer 1b/c AS oligonucleotides significantly attenuated the secondary mechanical allodynia on days 2 to 5 and significantly reduced thermal hyperalgesia on days 3 to 4 after injection. Injection of the missense oligonucleotides produced no effect on secondary mechanical allodynia and thermal hyperalgesia. Two-way analysis of variance showed a statistically significant difference between the AS and missense groups (P < 0.05; Figs. 5A and 6A).

Figure 5

Figure 5

Figure 6

Figure 6

Furthermore, the effects of 2 doses of Homer 1b/c AS oligonucleotides on secondary mechanical and thermal hypersensitization were examined. The higher dose (5 μg) of Homer 1b/c AS oligonucleotide significantly attenuated secondary mechanical allodynia and thermal hyperalgesia, but the lower dose (2.5 μg) had no effect. Compared with the saline group, both the behavioral response to the von Frey filament and heat stimuli were significantly reduced by administration of 5 μg AS oligonucleotides on day 4 (P < 0.05; Figs. 5B and 6B). No significant difference was found between the saline group and the 2.5-μg AS oligonucleotide group.

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Effect of Homer 1b/c AS Oligonucleotides on Pain Behavioral Responses and Locomotor Function in Naïve Rats

To examine whether Homer 1b/c AS oligonucleotides influenced the behavioral responses to mechanical and thermal stimulation in the animals without inflammation, we measured paw withdrawal thresholds and withdrawal latencies in naïve rats after Homer 1b/c AS oligonucleotide administration. The baseline responses to mechanical and thermal stimulation were determined as a pre-AS control. Homer 1b/c AS oligonucleotides (10 μg/10 μL) were administered intrathecally every 24 hours for 4 days. There was no significant change in the paw withdrawal thresholds or latencies after 4 days of AS oligonucleotide application (Fig. 7).

Figure 7

Figure 7

To evaluate the effect of Homer 1b/c deficiency on locomotor function, we examined locomotor function in naïve rats before and after oligonucleotide administration. The locomotor testing included placing, grasping, and righting reflexes. As shown in Table 1, the AS oligonucleotides (10 μg) failed to produce a significant effect on locomotor function. Convulsions and hypermobility were not observed after the administration of AS oligonucleotides. In addition, there were no significant differences in the general behaviors and spontaneous activity between the pre-AS and post-AS animals.

Table 1

Table 1

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DISCUSSION

The regional expression and function of Homer 1b/c in the mammalian central nervous system have been investigated using a variety of experimental approaches. In the brain, high levels of Homer 1b/c proteins are expressed constitutively in neurons, and they preferentially colocalize with mGluR5.1719 In rat striatal neurons, the disruption of mGluR5/Homer 1b/c binding with a cell permeable Tat-fusion peptide or the selective inhibition of Homer 1b/c synthesis with siRNAs reduced mGluR5 agonist-induced extracellular signal-regulated kinases (ERK)1/2 phosphorylation.20 Deletion of Homer 1 in mice resulted in an increased sensitivity to cocaine-induced locomotion and conditioned reward, augmented extracellular glutamate in the nucleus accumbens, and caused neurochemical abnormalities that are consistent with animal models of schizophrenia.2123 In the spinal cord, Homer 1b/c mRNA and protein are selectively distributed in the superficial dorsal horn,7,24,25 which was confirmed in the present study both by immunoblotting and immunohistochemistry. Because of the pivotal role of the superficial dorsal horn in the processing and transmission of peripheral noxious stimulation, the area-specific expression and distribution of Homer 1b/c in the spinal cord may have important implications toward the mechanisms of nociceptive processing.

AS technology presents a useful strategy for suppressing protein synthesis and has gained acceptance in the study of cell signaling and neuronal function.2628 AS oligonucleotides approaches have been used to address the roles of PSD-95 (another scaffolding protein in the PSD) in neuropathic pain and Homer 1b/c in cocaine-induced behavioral plasticity.4,29 In the current study, to elucidate the role of Homer 1b/c in chronic inflammatory pain, an AS oligonucleotide with previously demonstrated specificity against Homer 1b/c was used to downregulate the expression of Homer 1b/c in the spinal cord.

The present study revealed that monoarthritis induced by CFA resulted in upregulation of Homer 1b/c expression in the ipsilateral spinal dorsal horn. Spinal Homer 1b/c deficiency attenuated both the upregulation of Homer 1b/c expression and CFA-induced secondary mechanical allodynia and thermal hyperalgesia without affecting locomotor activities and baseline responses to noxious mechanical or thermal stimulation in rats. Previous studies have demonstrated that the identical Homer 1b/c AS oligonucleotide used in the present study suppressed the expression of Homer 1b/c, but did not affect the expression of the N-methyl-D-aspartate receptor subunits NR2A/2B, neuronal nitric oxide synthase, or SAP102 in rats.4 These results suggest that the effects of Homer 1b/c AS oligonucleotides are unlikely to be associated with changes in the expression of those proteins. Taken together, Homer 1b/c AS oligonucleotide–induced attenuation of secondary inflammatory pain seems to be attributable to direct and selective interference of the AS oligonucleotide on the Homer 1b/c mRNA transcript. Homer 1b/c protein production can be blocked when the Homer 1b/c AS oligonucleotide binds to the Homer 1b/c mRNA. It is conceivable that spinal Homer 1b/c has an important role in the maintenance of secondary hyperalgesia from chronic inflammation.

The precise mechanism underlying the involvement of Homer 1b/c in secondary hyperalgesia from chronic inflammation remains unclear. Homer 1b/c is a scaffold protein that contains an N-terminal EVH1 domain, which is capable of binding complementary motifs found in group I mGluRs, inositol 1,4,5-triphosphate (IP3) receptors, and Shank at synapses.17,30 Homer 1b/c seems to be involved in synaptic targeting and the localization of receptors, and/or receptor signaling. Emerging evidence indicates that the coordinated interaction of group I mGluRs with adaptor Homer proteins contributes to many functions, such as the membrane trafficking of mGluR1/5,3137 the coupling of mGluR1/5 with IP3 receptors,38 the signaling from mGluRs to N-type Ca2+ and M-type K+ channels,39 and the development of spines, axons, and synapses.4043 Homer 1b/c protein deficiency in the nucleus accumbens was shown to prevent the development of sensitization to repeated cocaine treatment.4 However, in the spinal dorsal horn, Homer 1a gene expression was transiently upregulated,24,25 and Homer 1b/c expression increased after loose ligation of the sciatic nerve in rats.7 Taken together, these results suggest that deficiency of spinal cord Homer 1b/c protein may alter mGluR function but not its expression or distribution in the spinal cord. Given the well-established role of group I mGluRs in the development or maintenance of inflammatory pain,4447 the AS oligonucleotide–induced reduction of inflammatory pain observed in the current study may result from the Homer 1b/c knockdown inducing a decrease in mGluR function. However, we cannot exclude other possibilities, such as changes in the synaptic localization of mGluRs and in the downstream signaling of mGluRs, particularly the mGluR-IP3/Ca2+ signaling pathway.

In conclusion, the present study demonstrated for the first time that intrathecal administration of Homer 1b/c AS oligonucleotides dose dependently attenuated the secondary mechanical allodynia and thermal hyperalgesia during the maintenance of CFA-induced monoarthritis. The AS oligonucleotides did not influence the behavioral baseline and locomotor function of the experimental animals. Our results indicate that the deficiency of Homer 1b/c protein in the spinal cord attenuates CFA-induced secondary mechanical and thermal hyperalgesia during the maintenance of chronic inflammatory pain. Thus, the present findings suggest that Homer 1b/c may be involved in the central mechanisms of chronic monoarthritis and provide novel therapeutic implications.

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DISCLOSURES

Name: Yong-Xing Yao, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Yong-Xing Yao has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Zhen Jiang, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Zhen Jiang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Zhi-Qi Zhao, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Zhi-Qi Zhao has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Tony L. Yaksh, PhD.

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ACKNOWLEDGMENTS

The authors thank Professor Yu-Qiu Zhang for her suggestions in designing the experiments, and Pei-Fen Wang and Hong-Ying Shan for their technical assistance. The authors thank Medjaden Bioscience Limited for assisting in the preparation of the manuscript.

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