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Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A2AR) decrease pro-inflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain, and that IL-10 can suppress such pain, we evaluated the effects of intrathecally (i.t.) administered A2AR agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single i.t. injection of the A2AR agonists ATL313 or CGS21680, 10-14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 wk. Neither drug altered the nociceptive responses of sham-operated controls. An A2AR antagonist (ZM241385) co-administered i.t. with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia, but had no effect on allodynia in the absence of the A2AR agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4-L6 spinal cord segments both 1 wk and 4 wk after a single i.t. ATL313 administration. Neutralizing IL-10 antibodies administered i.t. transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A2ARs following i.t. administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS.
Neuropathic pain, resulting from nerve injury or inflammation, affects approximately 4 million people in the USA alone (Taylor, 2006) and remains poorly managed by currently available therapeutics. Most of these therapeutics specifically target neurons. However, spinal glia (astrocytes and microglia) play an important role in facilitating and maintaining neuropathic pain in animal models (Watkins et al., 2007). Following the initial injury or inflammation, neuronal central sensitization occurs and normally surveying microglial cells become activated to a reactive state (Hanisch and Kettenmann, 2007). Activated glial cells release pro-inflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α), chemokines and other inflammatory mediators such as prostaglandins, reactive oxygen species and nitric oxide, contribute to the maintenance of central sensitization (Watkins et al., 2007). Recent studies have identified that decreasing spinal pro-inflammatory cytokines or increasing anti-inflammatory cytokines is effective in attenuating neuropathy-induced allodynia (DeLeo and Yezierski, 2001; Milligan et al., 2006; Watkins et al., 2007). An ideal pharmacological treatment for neuropathic pain would be to avoid short-term blockade of the downstream effects of glial activation and neuronal hyperexcitability, and instead “reset” activated glia back to their basal, surveying state or to an alternatively activated anti-inflammatory state (Gordon, 2003). As yet, no candidate drug has been identified that induces such changes.
One potential candidate for such a drug may be an agonist at a select adenosine receptor subtype. Adenosine can bind four different receptors: A1R, A2AR, A2BR and A3R. Most work investigating the effects of adenosine in pain models have used adenosine or non-selective agonists and antagonists, which will target multiple adenosine receptors. In addition, some studies have explored the effect of A1R agonists, as A1Rs are found predominantly on neurons (Hasko et al., 2007) and A1R agonists are antinociceptive in a number of different pain models (Lee and Yaksh, 1996; Yamamoto et al., 2003; Zahn et al., 2007).
A2AR agonists may be of special interest. A growing body of literature is presenting A2AR agonists as having potent anti-inflammatory effects on peripheral immune cells, including suppression of proinflammatory cytokines and enhanced production of the anti-inflammatory cytokine, interleukin-10 (IL-10) (Hasko and Cronstein, 2004). Such a pattern is consistent with A2ARs being Gαs-linked receptors that stimulate adenylyl cyclase resulting in increased cyclic adenosine monophosphate (cAMP) production (Hasko et al., 2007). In addition to peripheral immune cells, A2AR are found on a wide variety of cell types within the central nervous system (Dare et al., 2007). Although one cannot rule out the possibility of A2AR agonists exerting at least some of their effects on neurons, microglia are the surveying immunocompetent macrophages of the central nervous system, and astrocytes have immunogenic properties (Ren and Dubner, 2008). Therefore, it is possible that A2AR activation on microglia and astrocytes may produce anti-inflammatory effects within the spinal cord, thus alleviating allodynia from chronic pain states. The present series of studies was designed to explore this possibility through the use of A2ARagonists.
Pathogen-free male Sprague-Dawley rats (325-350 g: Harlan Laboratories, Madison, WI, USA) were used for all experiments. Rats were housed two per cage with standard rat chow and water ad libitum. Housing was in a temperature controlled environment (23 ± 2 °C) with a 12:12 light:dark cycle (lights on at 07:00). All procedures occurred in the light phase. All animals were allowed 1 wk of acclimation to the colony rooms before experimentation. The Institutional Animal Care and Use Committee of the University of Colorado at Boulder approved all procedures.
The A2AR agonist, ATL313 was a gift from PGxHealth, A Division of Clinical Data, Inc. (Charlottesville, VA, USA). The half-life of ATL313 is less than 30 min (Moore et al., 2008). The A2A agonist, CGS21680, and the mu (naloxonazine), kappa (nor-binaltrophimine) and delta (naltrindole) selective opioid receptor antagonists were purchased from Sigma (St. Louis, MO, USA). The A2AR selective antagonist, ZM24385, was purchased from Tocris Biosciences (Ellisville, MO, USA). All of the adenosine agonists and antagonists were dissolved in DMSO to create 10 mM stock concentrations and stored at −20 °C. Fresh aliquots were diluted to the appropriate concentration in sterile endotoxin-free isotonic saline (Abbot Laboratories, North Chicago, IL, USA). The opioid antagonists were made fresh immediately before injections. The vehicle for the adenosine agonists and antagonists was 0.01% DMSO saline solution given the dilution of the drugs form stock was 1:10 000 to yield a 1 μM dose. The vehicle for the opioid antagonists was 0.9% saline. All vehicle injections were administered equivolume to the drugs being tested. Rat IL-10 neutralizing antibodies were raised in sheep at the National Institute of Biological Standards and Control (South Mimms, Hertfordshire, UK) and purified by Avigen (Alameda, CA, USA). Normal sheep IgG was used as a control (Sigma, St Louis, MO, USA).
Rats were habituated to the testing apparatus for four consecutive days before testing. The von Frey test was performed on the plantar surface of each hind paw within the region of sciatic nerve innervation, as described previously (Milligan et al., 2000). A logarithmic series of 10 calibrated Semmes-Weinstein monofilaments (Stoelting, Wood Dale, IL, USA) were sequentially applied (from low to high intensity threshold) to the left and right hind paws in random order, each for 8 s at constant pressure to determine the stimulus intensity threshold stiffness required to elicit a paw withdrawal response. Log stiffness of the hairs is determined by log10 (milligrams × 10). The range of monofilaments used in these experiments (0.407-15.136 gm) produces a logarithmically graded slope when interpolating a 50% response threshold of stimulus intensity (expressed as log10 (milligrams × 10)(Chaplan et al., 1994). The stimulus intensity threshold to elicit a paw withdrawal response was used to calculate the 50% paw withdrawal threshold (absolute threshold) using the maximum likelihood fit method to fit a Gaussian integral psychometric function (Harvey, 1986). This method normalizes the withdrawal threshold for parametric analyses (Harvey, 1986). The behavioral testing was performed blind with respect to the drug administration.
Thresholds for behavioral response to heat stimuli applied separately to the tail and each hind paw were assessed using a modified Hargreaves test (Hargreaves et al., 1988). Baseline withdrawal values were calculated from an average of three consecutive withdrawal latencies of the tail, left and right hind paws. A cut-off time of 20 s was imposed to avoid tissue damage. As with the von Frey test, this behavioral testing was performed blind with respect to the drug administration.
Chronic constriction injury (CCI) (Bennett and Xie, 1988) of the left sciatic nerve was aseptically performed under isoflurane anesthesia. Four ligatures of 4-0 chromic gut were tied loosely around the left sciatic nerve at the level of the mid-thigh, as described previously (Milligan et al., 2004).
Animals were lightly anesthetized with isoflurane. The lumbar region was shaved and cleaned. An 18-gauge guide needle, with the hub removed, was inserted into the L5/6 intervertebral space. A PE-10 catheter was inserted into the guide needle, pre-marked such that the proximal end of the PE-10 tubing rested over the L4-6 lumbar spinal cord. All drugs were administered over 20 s (1 μl drug followed by 2 μl sterile saline flush) with a 30 s delay before removing the catheter and guide needle. Each animal was anesthetized for a maximum of 5 min and none incurred observable neurological damage from the procedure.
0.6 mm pieces of the L4-6 lumbar spinal cord were sectioned (20 μm), mounted onto gelatin-subbed slides, and treated with 0.03% H2O2 in Tris-Buffered saline for 15 min. Sections were incubated overnight in primary antibodies for monoclonal mouse anti-rat OX-42 (1:100, Pharmingen, San Diego, CA, USA) and monoclonal mouse anti-rat glial fibrillary acidic protein (GFAP; 1:100, ImmunO, Solon, Ohio, USA). After washing, the sections were incubated with biotinylated secondary antibodies (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. The sections were washed followed by 2 h incubation in Avidin-Biotin Complex (ABC, 1:400 Vector Laboratories, Burlingame, CA, USA), washed and reacted with 3, 3′ diaminobenzadine tetrahydrochloride (Sigma, St Louis, MO, USA). Glucose oxidase and D-glucose were used to generate hydrogen peroxide. Nickeleous ammonium sulfate was used with the DAB reaction to optimize the reaction product. Sections were dried overnight and then dehydrated with graded alcohol, cleared in histoclear and cover slipped with permount. From each animal's spinal cord, 5-7 sections within the L4-6 region were included in the analysis. The ipsilateral dorsal horn to the side of injury, of each section, was captured at 10x magnification as a tiff file. Each image was analyzed, under blinded conditions, using NIH image J using a gray scale. The signal pixels within the dorsal horn were identified above 3.5 standard deviations beyond a control region (lateral column of the cord). The integrated densitometry was calculated as the number of pixels and the average gray scales above the set background (Chacur et al., 2004).
Total RNA from the lumbar spinal cord was extracted using the standard phenol:chloroform extraction with TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's guidelines. Samples were treated with DNase to remove any contaminating DNA (Ambion, Austin, TX). Total RNA was reverse transcribed into cDNA using Superscript II First-Strand Synthesis System (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized using total RNA, random hexamer primer (5 ng/μl) and 1mM dNTP mix (Invitrogen, Carlsbad, CA) and incubated at 65 °C for 5 min. Following 2 min incubation on ice, a cDNA synthesis buffer (5 X RT buffer, Invitrogen, Carlsbad, CA) and dithiothreitol (10 mM) was added and incubated at 25 °C for 2 min. Reverse transcriptase (Superscript III, 200 Units, Invitrogen, Carlsbad, CA) was added to a total volume of 20 μl and incubated for 10 min at 25 °C, 50 min at 42 °C and deactivating the enzyme at 70 °C for 15 min. cDNA was diluted 2-fold in nuclease-free water and stored at −80 °C. The cells within the CSF were processed into cDNA using a Cells-Direct III cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer's guidelines. Briefly, after washing the cells in 100 μl Dulbecco's phophate buffered saline, the CSF cells were lysed in 11 μl lysis buffer and lysis solution for 10 min on ice. 10 μl of cell lysate was added to 1μl RNase inhibitor and incubated at 75 °C for 10 min. First-strand cDNA was synthesized by adding 2 μl oligo (Dt), 1 μl dNTP and 7.8 μl nuclease free water to the cell lysate and incubating at 70 °C for 5 min. Following 2 min on ice, 6 μl 5 x RT buffer, 1 μl RNAse inhibitor, 1 μl Superscript III, and 1 μl dithiothreitol was added to the cell lysate and incubated at 50 °C for 50 min, 5 min for 85 °C. Finally 1 μl RNase H was added and incubated at 37 °C for 20 min. All cDNA was stored at −80 °C until RT-PCR was performed.
Primer sequences were obtained from the Genbank at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov) and displayed in Table 1. Primers were generated to span an intron to eliminate genomic interference in the CSF samples that did not receive DNAse treatment. The CSF samples were approached in this fashion as the RNA isolation through to cDNA synthesis is completed in the same tube. Therefore, genomic variability was minimized by not doing a DNase treatment but rather designing primer sequences that spanned an intron. Amplification of the cDNA was performed, in a blinded procedure, using Quantitect SYBR Green PCR kit (Qiagen, Valenica, CA) in iCycler iQ 96-well PCR plates (Bio-Rad, Hercules, CA) on a MyiQ single Color Real-Time PCR Detection System (Bio-Rad). The reaction mixture (26 μl) was composed of QuantiTect SYBR Green (containing fluorescent dye SYBR Green I, 2.5 mM MgCl2, dNTP mix and Hotstart Taq Polymerase), 10 nM fluorescein, 500 nM of each forward and reverse primer (Invitrogen, Carslbad, CA), nuclease-free water and 1 μl of cDNA from each sample. Each sample was measured in duplicate. The reactions were initiated with a hot start at 95 °C for 25 min, followed by 40 cycles of 15 s at 94 °C (denaturation), 30 s at 55-60 °C (annealing) and 30 s at 72 °C (extension). Melt curve analyses were conducted to assess uniformity of product formation, primer-dimer formation and amplification of non-specific products. The PCR product was monitored in real-time, using the SYBR Green I fluorescence, using the MyiQ single Color Real-Time PCR Detection System (Bio-Rad). Threshold for detection of PCR product was set in the log-linear phase of amplification and the threshold cycle (CT) was determined for each reaction. The level of the target mRNA was quantified relative to the housekeeping gene (β-actin) using the comparative CT(ΔCT) method. The expression of β-actin was not significantly different between treatments.
Behavioral measures were normalized as described above and analyzed using repeated measures 2-way ANOVA with time and treatment as main effects. The integrated density from the histology, RT-PCR data was analyzed using a two-way ANOVA with surgery and drug administration as main effects. Bonferroni post-hoc tests were used where appropriate and P < 0.05 was considered statistically significant. For ease of reading, the basic statistical values are shown in the text while the more extensive statistical information can be found in the figure legends.
Baseline behavioral measures were recorded after 4 days of 40 min/day habituation to the testing environment. CCI or sham surgery was then conducted and behavioral responses to mechanical stimuli or thermal stimuli were tested, in separate groups of rats, at 4 and 10 days after surgery. At 10-14 days after surgery, an acute i.t. administration of ATL313 (0.1 μM or 1 μM in 1 μl), CGS21680 (1 μM or 10 μM in 1μl ) or equivolume vehicle was given (n = 6-7 rats per group) in groups tested for mechanical allodynia. Based on these results, an acute i.t. administration of either ATL313 (1 μM) or vehicle was given to CCI and sham groups tested for thermal hyperalgesia. For both the mechanical and thermal testing, behavioral responses were measured 4 h, 24 h, 72 h, and weekly for 6 wk after i.t. drug administration.
As in Experiment 1, baseline behavioral measures were performed after 4 days of 40 min/day habituation to the testing environment. CCI was conducted and behavioral responses to mechanical stimuli were tested 4 and 10 days after surgery. At 10-14 days after surgery, an acute i.t. administration of ATL313 (1 μM) or vehicle (1 μl) was given with either ZM241385 (10 μM; A2AR antagonist) or vehicle (1 μl; n = 6 rats per group). A ten-fold higher dose of the antagonist was used to ensure complete blockade of A2ARs. Behavioral responses were measured 1, 2, 3, 4, 6 and 24 h after drug administration. In a second group of animals, CCI surgery and behavioral measures were conducted as described above. At 10-14 days after surgery, ATL313 (1 μM) was administered i.t. One week following ATL313 administration, ZM21385 (10 μM) or equivolume vehicle (1 μl) was administered i.t. Behavioral responses were measured 24 h after ATL313 administration, before ZM241385 and 1, 2, 3, 4, 6 and 24 h after drug administration.
CCI surgery and behavioral measures were conducted as described in Experiment 1. At 10-14 days after surgery, a single i.t. injection of ATL313 (1 μM) or equivolume vehicle was administered. The rats then received additional injections, the same as that received on the first injection, once every four weeks after the first injection for a total of three injections. The behavioral testing was conducted before surgery, before each injection, 24 h after each injection and weekly thereafter for 14 wk after the first injection (n = 6/CCI group and n = 6/sham group).
As previous reports have implicated endogenous opioids in some A2AR-mediated effects at supraspinal sites (Schiffmann et al., 2007), a cocktail of selective opioid receptor antagonists was used to explore whether endogenous opioids account for the reversal of CCI-induced allodynia by ATL313. CCI surgery and behavioral measures were conducted as described in Experiment 2. At 10-14 days after surgery ATL313 (1 μM) was administered i.t. Once the CCI-induced mechanical allodynia was stably reversed by ATL313, a combination of selective opioid receptor antagonists: mu (Naloxonazine, 1 μM in 1 μl), kappa (nor-Binaltrophimine, 1 μM in 1 μl) and delta (Naltrindole, 1 μM in 1 μl) or equivolume vehicle (3 μl) was administered i.t. (n = 6/group). These doses were chosen to ensure adequate treatment, as they are each ten-fold higher than those known to be effective in abolishing the effects of the opioid agonists in vivo(Lu et al., 2004; Nielsen et al., 2007). Behavioral responses were measured 24 h after ATL313 administration, before opioid antagonist/vehicle administration and 0.5 h, 1 h, 2 h and 3 h after drug administration.
Chronic constriction injury was conducted and behavioral responses to mechanical stimuli were tested as described in Experiment 2. At 10-14 days after surgery, an acute i.t. administration of ATL313 (1 μM) or equivolume vehicle (1 μl) was coadministered with either sheep anti-rat neutralizing IL-10 IgG antibodies (0.2 μg/ml, 10 μl) or equivolume and equidose sheep IgG (0.2 μg/ml, 10 μl) was co-administered (n = 6 rats per group). Behavioral responses were measured 3 h, 6 h, 24 h, 48 h and 1 wk after drug administration. A second injection of sheep anti-rat neutralizing IL-10 IgG antibodies (0.2 μg/ml, 10 μl) or equivolume and equidose sheep IgG was injected i.t. one week later and behavioral responses to mechanical stimuli were measured before drug administration and 1 h, 2 h, 3 h, 4 h, 6 h, 24 h 48 h and 1 wk after drug administration.
Groups of rats received either CCI or sham surgery, followed 10-14 days after surgery with either ATL313 (1 μM) or equivolume vehicle i.t. One week and four weeks after drug administration, the animals were injected intraperitoneally with a terminal dose of sodium pentobarbital (n = 3-4/group). Animals were transcardially perfused with ice-cold heparinized saline followed by 4% paraformaldehyde/0.1M phosphate buffered saline. Lumbar spinal cord sections were dissected and post-fixed overnight in 4% paraformaldehyde. The lumbar spinal cord was cryoprotected in 30% sucrose solution and processed for microglial and astrocytes activation using immunohistochemistry.
Groups of animals (n=8-10/group) received either CCI or sham surgery, followed 10-14 days after surgery with either ATL313 (1 μM) or equivolume vehicle i.t. One week after drug administration, rats were deeply anesthetized with sodium pentobarbital (intraperitoneal injection, 0.8 ml). CSF was aspirated via acute lumbar technique as described for the acute lumbar i.t. injections. The CSF was centrifuged at 1000 × g for 10 min at 4 °C to pellet the cells. The CSF cells were processed into cDNA as described above. Animals were then transcardially perfused with ice-cold saline for 2 min. The lumbar spinal cord (L4-6 region) was dissected out. The meninges and lumbar tissue were processed together for gene expression. In a separate group of animals, the overlying meninges were separated from the spinal tissue and processed for gene expression. The CSF was collected on both groups of animals for mRNA analysis. All tissues were flash frozen in liquid nitrogen and stored at −80 °C until further analysis.
We examined the effects of two structurally different A2AR agonists on CCI-induced mechanical allodynia in order to identify whether i.t. administration of A2AR agonists produces comparable results to that observed in vitro (Hasko et al., 1996). Figure 1 shows that the mechanical allodynia induced following CCI surgery remains stable from 10 d throughout the duration of the study (8 wk after surgery). A single bolus dose of ATL313 (1 μM), administered i.t. between 10 and 14 d after surgery, induced a significant attenuation of the mechanical allodynia induced by CCI surgery for 4 wk in both the ipsilateral and contralateral hindpaw (P < 0.01, interaction: F9,160 = 2.176, n = 6-7/group). Sham surgery had no significant impact on behavior throughout the study. Additionally, ATL313 (1 μM) had no significant effect on behavioral responses of sham-operated rats (P > 0.05, Figure 1). The lower dose of ATL313 (0.1 μM) administered to allodynic rats, had no significant effect on the mechanical allodynia at any time tested (P > 0.05, main effect of drug F1,9 = 0.025, Figure 1).
Based on these results, the effect of 1 μM ATL313 was tested for its effects on CCI-induced thermal hyperalgesia (Figure 2). Comparable results were obtained against thermal hyperalgesia as that against mechanical allodynia; that is, ATL313 significantly reversed CCI-induced thermal hyperalgesia for 4+ wk, with no effect of the drug on the responses of sham controls (P < 0.0001, interaction: F27,200 = 11.04, n = 6/group).
To begin to provide converging lines of evidence that A2AR agonism underlies the effects observed above, a second, structurally distinct A2AR agonist, CGS21680, was also tested and found to produce the same effect as that of ATL313 in allodynic rats at a ten-fold higher dose (P < 0.001, interaction: F9,160 = 2.865, n = 6/group, Figure 3), which is consistent with their relative receptor binding affinities. Therefore, both ATL313 and CGS21680 produced a reduction of neuropathic-induced allodynia for 4+ wk after a single i.t. administration.
As a second test to validate that the effect of ATL313 is indeed A2AR mediated, a single i.t. injection of an A2AR selective antagonist (ZM23185, Table 1) was co-administered with a single i.t. injection of ATL313 10-14 d after CCI surgery. Co-administration of the A2AR agonist (ATL313) and A2AR antagonist (ZM23185, 10 μM) abolished the effect of the A2AR agonist alone in reversing neuropathic-induced allodynia (Figure 4A, P < 0.001, Interaction: F6,93 = 12.67, n = 6/group). The A2AR antagonist (ZM23185) administered alone produced no effect on CCI-induced allodynia (P > 0.05). Therefore, the initiation of the effect of an A2AR agonist on neuropathic pain is mediated by A2ARs. In order to assess whether the sustained effect is also mediated by A2AR, we administered the A2AR antagonist 1 wk after the administration of the A2AR agonist (ATL313). There was no significant effect of the A2AR antagonist, ZM23185, on the established ATL313-induced reversal of CCI-induced allodynia (P = 0.76, main effect of drug: F1,9 =0.696, n = 6/group, Figure 4B).
In order to assess whether tolerance to the A2AR agonist would occur with repeated dosing, we gave the A2AR agonist once every 4 wk, when the drug effect was still apparent (Figure 5). The rats did not develop tolerance to ATL313 when given once every 4 wk as the reversal of allodynia was maintained for 11 wk after the first injection, compared to CCI+vehicle (P < 0.0001, interaction: F39,260 = 6.439, n = 6/group). The reversal of allodynia may have lasted longer except that the allodynia induced by the CCI was resolving by 11 wk after intrathecal injection.
Given the interaction between adenosine receptors and opioid receptors, we assessed whether the long term reduction in allodynia following ATL313 administration in neuropathic rats was opioid receptor mediated. To ensure that we blocked all opioid receptors effectively, we used a ten-fold higher dose than that used previously and used antagonists for mu, kappa and delta opioid receptors. After i.t. ATL313 stably reversed CCI-induced mechanical allodynia for 1 wk, a combination of kappa (1 μM nor-binaltrophimine), delta (1 μM naltrindole) and mu (1 μM naloxonazine ) opioid receptor antagonists were co-administered i.t. Before administration of the opioid antagonists or vehicle, there was no significant difference between the groups (Figure 6). After the administration of the opioid antagonists, there was no significant effect of the opioid antagonists on the A2AR-mediated reversal of neuropathic allodynia compared to vehicle injections (P = 0.73, main effect of drug: F1,7 = 0.120, n = 6/group). Therefore, while we have not tested in involvement of opioids in the onset of the A2AR drug effect, the sustained A2AR reversal of mechanical allodynia does not appear to involve endogenous opioids.
Previous studies of A2AR effects on peripheral immune cells have implicated increased release of IL-10 as importantly contributing to the anti-inflammatory effects of A2AR agonists (Hasko et al., 1996; Csoka et al., 2007b). No prior studies have explored whether IL-10 may be involved in A2AR effects centrally. Therefore, we assessed whether neutralizing IL-10 in the intrathecal space, using IL-10 antibodies, would abolish the effect of the A2AR agonist. After establishment of CCI-induced mechanical allodynia, we administered i.t. ATL313 together with neutralizing IL-10 IgG versus control IgG (Figure 7). There was no significant effect of the IL-10 antibodies when co-administered with the ATL313, suggesting that the initial effect of the A2AR agonist is not via IL-10 (P = 0.314, main effect of drug: F3,20 = 1.263). One wk following the first i.t. injection, the rats were injected with a second dose each of either neutralizing IL-10 IgG or control IgG, according to the same grouping as the first injection. Now, neutralizing IL-10 reversed the enduring effect of the A2AR agonist (P < 0.05, main effect of drug: F3,20 = 4.001, n = 6/group). Interestingly, the effect of the A2AR agonist returned by 48 h after the neutralizing IL-10 antibodies had been administered, suggesting that the A2AR agonist induces sustained release of IL-10 contributing to the long-lasting effect of the drug. However, given the rats received the IL-10 neutralizing antibodies in both the first and second injection, it is not certain whether the first injection of IL-10 neutralizing antibody may have potentially impacted the effect of the second.
Previous studies of peripheral immune cells have documented that A2AR agonists can suppress the expression of activation markers on monocytes/macrophages (Mantovani et al., 2002; Kreckler et al., 2006; Hasko et al., 2007). No prior studies have explored whether similar effects could be produced centrally. Therefore, we assessed the effect of ATL313 on the predominant immunocompetent cells within the central nervous system by measuring immunohistochemical indices of glial activation, known to occur in spinal cord dorsal horn, in both the ipsilateral and contralateral sides, in response to CCI (Figures (Figures88 & 9). On the ipsilateral side, microglial activation (measured by OX-42 labeling) in CCI rats was significantly lower 1 wk (P < 0.001, F3,9 = 14.85) and 4 wk (P < 0.01, F3,9 = 7.37) after a single i.t. dose of ATL313, compared to rats receiving CCI + vehicle, sham surgery + vehicle, or sham surgery + ATL313. In addition, there was a significant attenuation of the astrocyte activation marker, GFAP, on the ipsilateral side, in CCI rats at 1 wk (P < 0.01, F3,11 = 8.32) and 4 wk (P < 0.05, F3,12 = 4.14) after a single i.t. dose of ATL313, compared to rats receiving CCI + vehicle. On the contralateral side, there was no significant differences in microglial activation markers between any of the groups at either 1 wk or 4 wk after drug administration (F3,8 = 2.090, P = 0.18). Astrocyte activation marker, GFAP, was significantly increased in the contralateral dorsal horn following CCI surgery compared to sham+ATL313 at 1 wk (P < 0.05) and both sham controls at 4 wk (P < 0.05). The increase in astrocytes activation observed in CCI was significantly attenuated following administration of ATL313 at both 1 wk (P < 0.001, F3,11 = 13.96) and 4 wk (P< 0.01, F3,10 = 10.28).
Previous studies of peripheral immune cells have documented that A2A agonists can increase IL-10 gene expression and suppress the pro-inflammatory cytokine TNF-α gene expression in monocytes/macrophages (Hasko et al., 1996; Csoka et al., 2007a). No prior studies have explored whether similar effects could be produced centrally. Here, tissues (CSF cells, L4-L6 lumbar spinal cord with overlying meninges, or meninges alone, n = 6-8/group) were collected after stable reversal of CCI-induced mechanical allodynia by ATL313. As shown in Figure 10, there was a significant increase in IL-10 gene expression in cells within the CSF (2-way ANOVA with surgery and drug as main effects, drug effect: P < 0.05, F1,22 = 159.5). There was no significant difference in IL-10 mRNA in the meninges between groups (drug effect: P = 0.33, F1,22 = 0.976) or in the lumbar tissue with the overlying meninges (drug effect: P=0.898, F1,22 = 0.017), suggestive that the elevated IL-10 gene expression is occurring in cells resident within the meninges or infiltrating from the periphery.
There was a significant decrease in TNF-α gene expression in the CSF cells (2-way ANOVA, drug effect, P < 0.05, F1,22 = 4.591) but no significant difference between CCI and sham (P=0.58, F1,22 = 0.316) or interaction (P=0.58, F1,21 = 0.316). The TNF-α mRNA attenuation by ATL313 does support previous in vitro experiments showing suppression of TNF-α production in peripheral immune cells (Hasko et al., 1996). In addition, like IL-10 gene expression, there was no significant difference in TNF-α gene expression between any of the groups in the lumbar and meningeal tissue or the meningeal tissue alone (drug effect: lumbar and meninges: P = 0.832, F1,22 = 0.046, meninges: P = 0.306, F1,22 = 1.094).
This study documents that a single intrathecal bolus injection of A2AR agonists reverse neuropathic pain for 4+ wk. This unprecedented, enduring reversal occurred for both mechanical allodynia and thermal hyperalgesia. The A2AR agonist effects likely involve suppression of astrocyte and microglial activation based on activation marker analysis plus involvement of the anti-inflammatory cytokine IL-10 in the pain suppression as IL-10 receptors are expressed by spinal glia, but not spinal neurons (Ledeboer et al., 2003). The effects are not unique to one A2AR-selective compound, as comparable results were obtained using two structurally distinct A2AR-selective agonists (ATL313 and CGS21680). Whether ATL313's increased potency, relative to CGS21680, reflects differences in receptor affinity, tissue penetration, relative rates of degradation/clearance, or other differences is unknown. The A2AR agonists are not analgesic, but rather anti-allodynic/anti-hyperalgesic, as they have no effect on non-neuropathic animals. Further, the long duration A2AR agonist effect in neuropathic rats is not opioid-mediated, another positive feature for considering targeting A2AR for pain control. The attenuation of neuropathic pain is mediated by A2AR activation initially, but the sustained reversal of allodynia is likely mediated by IL-10 release, possibly from resident or recruited cells in the intrathecal space. The efficacy of A2AR-selective agonists in producing sustained reversal of neuropathic pain is not limited to the therapy being administered shortly (10 days) after neuropathic pain onset as equally potent, sustained reversal of neuropathic pain is observed when treatment is administered 6 wk after CCI as well(Loram et al., 2009).
To date, only gene therapy produces longer than ~1 day of pain relief from single injections. Even gene therapy is challenging in the intrathecal space, with single-dose viral vectors failing in efficacy by ~2 wk (Milligan et al., 2005a; Milligan et al., 2005b) and repeated injections of optimized gene therapies required for more extended pain resolution. Given the difficulties of bringing intrathecal gene therapy to clinical trials, identifying novel drug approaches for extended pain relief may be ideal. Also, once monthly intrathecal drugs that induce endogenous spinal IL-10 avoids potential peripheral immunosuppression inherent in daily systemic administration of glial activation inhibitors, as these may compromise the responses of peripheral immune cells as well (Watkins and Maier, 2003).
IL-10 gene therapy studies provide insights for the potential of single dose intrathecal A2AR agonists for long-term pain control. Foremost is that no tolerance develops with extended IL-10 exposure for resolving thermal hyperalgesia and allodynia. Optimized IL-10 gene therapy provides 3+ months of pain suppression and, when neuropathic pain does reoccur, an additional IL-10 gene therapy treatment again reverses neuropathic pain (Sloane et al., 2009b). Uncompromised pain reduction over extended time periods is recapitulated here where single intrathecal doses of ATL313 delivered once each month provided sustained, potent, suppression of neuropathic pain. In addition, IL-10 gene therapy operates by driving IL-10 production by cells within the CSF space, with IL-10 induced in this manner acting as a protracted intrathecal delivery of IL-10 to spinal cord glia, thereby suppressing pain (Sloane et al., 2009b). Upregulation of IL-10 in CSF cells appears likely to be recapitulated by single-dose intrathecal A2AR agonists, suggestive that this may well contribute to both suppression of spinally mediated neuropathic pain and glial activation.
Previous studies report that A2AR activation reduces neuroinflammatory symptoms in spinal cord injury, including motor deficits and markers of neuronal injury (Cassada et al., 2002; Reece et al., 2006). We have recently extended this to show that the A2AR agonist, CGS21680, also potently suppresses paralysis in a rat model of multiple sclerosis (Loram et al., 2009). Thus A2AR agonism has far broader potential clinical utility than neuropathic pain.
No previous reports identified that A2AR agonists elevate IL-10 in vivo, either peripherally or centrally, or that blocking IL-10 relieves symptoms, such as neuropathic pain. Unlike in vitro studies where IL-10 is measured within 24 h following 24 h of constant A2AR agonist exposure (Hasko et al., 1996; Kreckler et al., 2006; Csoka et al., 2007b), we have identified that the increase in IL-10 production is sustained in vivo following A2AR agonist administration in neuropathic animals for 1+ wk but most likely 4 wk given the duration of effect on neuropathic pain. Comparable to in vitro studies, it appears that an inflammatory stimulus is required, such as that seen following CCI surgery and subsequent neuropathy, in order for the A2AR agonist to potentiate the IL-10 production. While we did not identify a significant interaction between surgery and drug effect in the IL-10 mRNA, higher values were obtained in A2AR agonist-treated neuropathic rats compared to vehicle-treated neuropathic animals.
Adenosine modulation may reduce neuropathic pain via activation of adenosine receptors either/or within spinal cord or resident or recruited immunocompetent cells within meninges or CSF, (Ribeiro et al., 2002; Hasko and Cronstein, 2004; Dare et al., 2007). We have previously shown that meningeal cells can produce pro-inflammatory cytokines following activation both in vivo and in vitro(Wieseler-Frank et al., 2007). Also, CSF cells significantly increase following peripheral neuropathy and intrathecal IL-10 gene therapy, and IL-10 gene therapy shifts the cells from a predominantly ED1, or classically-activated macrophage phenotype, to that of ED2, the alternatively-activated or anti-inflammatory phenotype (Sloane et al., 2009b). Potentially, given increased IL-10 mRNA in CSF cells in response to A2AR agonists, such drugs may alter the status of the cells recruited to, or resident within, the CSF and/or meninges into alternatively activated macrophages producing anti-inflammatory IL-10, especially under neuroinflammatory circumstances. The observed glial suppression may reflect A2AR agonist diffusing into spinal tissue altering glial function. Alternatively, it is possible that downstream mediators produced within CSF following A2AR agonists can penetrate spinal tissue and affect glia. In both A2AR knockout mice, and in our study of A2AR agonists, neuropathic pain was attenuated, correlated with reduction in glial activation markers within the spinal cord (Bura et al., 2008). These seemingly contradictory findings may reflect that glial activation in knockout mice was attenuated by a reduction in peripheral A2AR activation, known to be pro-nociceptive (Taiwo and Levine, 1990; Doak and Sawynok, 1995; Khasar et al., 1995), thereby affecting inputs to the spinal cord. In contrast, in our study the glial activation was attenuated by direct A2AR action within the spinal cord or, more likely, diffusion of CSF-derived IL-10, suppressing glial activation.
Our data are unique in that a single intrathecal A2AR agonist injection produces remarkably enduring reversal of allodynia compared to prior reports where A2AR effects were only measured within the first 4 h (Lee and Yaksh, 1996; Poon and Sawynok, 1998; Yoon et al., 2005; Zahn et al., 2007), a time when our results show suboptimal impact on allodynia. Also, all previous A2AR agonist studies have used nanomolar doses, below our lowest dose of ATL313 (0.1 μM), a dose where we did not detect an effect. Given that the effects from A2AR activation take a few hours to achieve, timing of behavioral observations is critical. In a spinal nerve ligation model of neuropathic pain, intrathecal adenosine (non-selective adenosine receptor agonist) and an A1R agonist each attenuated allodynia for up to 24 h, but no observations were reported after that (Lavand'homme and Eisenach, 1999; Zahn et al., 2007). Studies using the non-selective agonist NECA suppressed thermal and tactile nociception in rats, dissipating within 1 h (DeLander and Hopkins, 1987). Systemic or intrathecal A1R agonists produce anti-hyperalgesia and anti-allodynia, resolving within 2 h (Reeve and Dickenson, 1995; Lee and Yaksh, 1996; Sawynok, 1998; Curros-Criado and Herrero, 2005). Therefore, it is possible that activating neuronal adenosine receptors is short-lived versus the prolonged resetting of glial and/or CSF/meningeal immune cells following activation of A2ARs. Reversal of allodynia, observed here, show that unlike IL-10 protein or IL-1 receptor antagonist (Milligan et al., 2005a; Ledeboer et al., 2007), which produce immediate effects, at least 4 h and possibly even 24 h is required to optimize the reversal of allodynia. This slow onset of reversal, and given that IL-10 neutralizing antibodies failed with co-administration with the A2AR agonist, suggests that downstream mediators, such as IL-10, requires time to develop and/or that immune cells need to be recruited and then phenotypically shifted to an anti-inflammatory profile before a full benefit can occur for neuropathic pain.
The potential impact of A2AR agonists as therapeutic agents has not yet been realized, at least for neuroinflammatory diseases (Yan et al., 2003), since too short a time course has been included or the in vivo models have limited the ability to identify the astounding duration of drug effect. We are currently investigating the effect of A2AR agonists on a model of multiple sclerosis (experimental autoimmune encephalitis) and revealing dramatic improvement of motor function following single intrathecal A2A agonist administration (Loram et al., 2009), comparable to that seen following pDNA-IL-10 (Sloane et al., 2009a). If such observations are any indication, A2AR agonists are potentially exciting candidates for the treatment of chronic pain and possibly other neuroinflammatory diseases.
Financial support for these studies was provided by PGxHealth, LLC, a Division of Clinical Data, Inc., the American Pain Society Future Leaders in Pain Management Small Grants Program, an International Association for the Study of Pain International Collaborative grant, American Australian Association Merck Company Foundation Fellowship, National Health and Medical Research Council CJ Martin Fellowship (ID 465423;M.R.H.) and by NIH grants DA02422 and DA017670. We thank Avigen for the purification of anti-IL10.