Anti-hyperalgesic and morphine-sparing actions of... : PAIN (original) (raw)
1 Introduction
Neuropathic pain, or chronic pain due to nerve injury, is a prevalent condition for which currently there is no effective treatment (Zimmermann, 2001). It is a condition that is often refractory to opioids or requires large doses that possess unacceptable side effects (Martin and Eisenach, 2001). Neuropathic pain usually is persistent, and nerve injury can produce sensory/motor deficits and other paradoxical sensations of a qualitative nature, such as hyperesthesias (heightened but non-painful appreciation of sensation), paresthesias (tingling and pickling sensations) and dysesthesias (unpleasant or painful sensation).
The pathophysiology of neuropathic pain has been investigated using various animal models. Both peripheral and central mechanisms contribute to the pathophysiology of neuropathic pain. There is compelling evidence demonstrating that hyperalgesia (exacerbated pain in response to noxious stimulation), allodynia (pain perceived in response to normally non-noxious stimuli), and ongoing pain associated with nerve injury involves participation of activated glia and cytokines. Products released by activated glia (including cytokines) contribute directly to the pathology of neuropathic pain (Watkins et al., 1994; Wagner et al., 1998; DeLeo and Colburn, 1999; Milligan et al., 2001). Proinflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α), are normally expressed in low concentrations in the spinal cord; however, this expression increases after peripheral nerve injury (Bethea et al., 1998; Sweitzer et al., 2001a,b) and inflammation (Bao et al., 2001). In the central nervous system (CNS), the main source for proinflammatory cytokines are activated glia (DeLeo and Yezierski, 2001; Watkins et al., 2001a,b) and inhibitors of glial activation have shown to prevent the development of nerve-injury and inflammation-induced hyperalgesia and allodynia (Meller et al., 1994; Watkins et al., 1997; Sweitzer et al., 2001c). Recently we have shown that glial activation and enhanced proinflammatory cytokines level in the lumbar spinal cord down regulates the morphine analgesia in nerve-injured rats (Raghavendra et al., 2002). In an earlier study, our laboratory showed that the glial modulator, propentofylline reversed the development of allodynia in L5-spinal nerve-transected rats, which directly correlated with its ability to suppress glial activation (Sweitzer et al., 2001c). Extending that study, we have now shown that chronic intrathecal treatment of propentofylline to L5 spinal nerve-transected rats prevents the development of hyperalgesia and activation of spinal proinflammatory immune responses. Further, propentofylline treatment spared the analgesic actions of acute morphine.
2 Materials and methods
2.1 Animals
Male Sprague–Dawley rats (Harlan, Indianapolis, IN, USA) weighing 175g–200 g at the start of surgery were used. The animals were allowed to habituate to the housing facilities for at least 1 week before the experiments were begun. Behavioral studies were carried out in a quiet room between the hours of 09:00 and 11:00. The Institutional Animal Care and Use Committee at Dartmouth College approved procedures in this study. Efforts were made to limit distress and to use the minimum number of animals necessary to achieve statistical significance as set forth by the International Society for the Study of Pain guidelines (Covino et al., 1980).
2.2 Surgery
The unilateral peripheral mononeuropathy was produced according to the method described earlier by Colburn et al. (1999). Briefly, rats were anesthetized with halothane in an O2 carrier (induction 4%, maintenance 2%). A small incision to the skin overlying L5–S1 was made followed by retraction of the paravertebral musculature from the vertebral transverse processes. The L6 transverse process was partially removed exposing the L4 and L5 spinal nerves. The L5 spinal nerve was identified, lifted slightly, and transected. The wound was irrigated with saline and closed in two layers with 3-0 polyester suture (facial plane) and surgical skin staples. Sham surgeries were identical minus the transection of the L5 nerve.
2.3 Behavioral tests
Mechanical and thermal nociceptive threshold was evaluated in sham and L5 spinal nerve-transected rats. Mechanical nociceptive thresholds were evaluated using an Analgesy-Meter (Ugo Basile, Comerio, Italy) as explained by Stein et al. (1990). Rats were gently held and incremental pressure (maximum 250 g) was applied onto the dorsal surface of the ipsilateral hind paw. The pressure required to elicit paw withdrawal, the paw pressure threshold (in grams), was determined. Thermal nociceptive thresholds were evaluated by the hot water tail-flick test (Bian et al., 1999), which consisted of immersing the tail in water maintained at 49 °C (maximum for 15-s) and recording the latency to a rapid flick.
2.4 Evaluation of the anti-hyperalgesic activity of propentofylline in neuropathic rats
Baseline threshold to noxious mechanical and thermal stimuli were established prior to the surgery. Propentofylline (Sigma, MO, USA; 0.1, 1 and 10 μg/rat) or saline (for control group of animals) was injected once daily intrathecally (i.t.) via lumbar puncture at the L4/5 level under brief halothane anesthesia (_n_=8/treatment). Drug administration on the day of surgery preceded the operation by 1 h. Daily administration was in the early evening and continued until day 10 of post-transection. Development of thermal and mechanical hyperalgesia was recorded on day 1, 3, 5, 7 and 10 post-transection. Propentofylline treatment preceded behavioral testing by 15 h. For accessing development of thermal and mechanical hyperalgesia, threshold latency (seconds in thermal test and grams in mechanical test paradigm) recorded on postoperative days 1, 3, 5, 7 and 10 were subtracted by preoperative (basal) latency, and was expressed as a relative decrease in nociceptive threshold.
2.5 Evaluation of the acute analgesic effect of morphine in sham-operated or nerve-injured rats treated chronically with propentofylline or saline
On postoperative day 11, both control and L5 spinal nerve-transected rats, which were chronically treated with propentofylline (10 μg, i.t.) or saline were administered morphine (Sigma) at 1–10 mg/kg, i.v. (via the tail vein). The analgesic activity of morphine in these rats was evaluated using the hot water tail-flick and paw-pressure Analgesy Meter. Analgesic effect was recorded 60 min after i.v. administration of morphine. The threshold response of rats to noxious stimuli prior to administration of morphine served as the basal latency and the analgesic effect was expressed as percent maximal possible effect (%MPE).
2.6 Qualitative assessment of glial fibrillary acidic protein (GFAP) and OX-42 immunoreactivity in L5 lumbar spinal cord
On day 11 after recording the analgesic effect of acute morphine, animals were anesthetized and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) (pH 7.4), followed by 4% paraformaldehyde in PBS. Lumbar spinal cord sections were harvested and processed as previously described (Colburn et al., 1999). Immunohistochemistry was performed on 20-μm free-floating L5 spinal cord sections. A monoclonal antibody to OX-42 (1:2 working dilution; William F. Hickey, Dartmouth Hitchcock Medical Center, Lebanon, NH) was used to label the expression of CR3/CD11b on activated microglia. A polyclonal antibody to GFAP (1:20,000 working dilution; Dako Corp., Carpinteria, CA) was used to label astrocytes.
2.7 Tissue collection for real-time reverse transcription–polymerase chain reaction (RT–PCR), RNase protection assay, and enzyme-linked immunosorbent assay (ELISA) analysis
To quantify glial fibrillary acidic protein (GFAP), macrophage antigen complex-1 (Mac-1) and cytokine mRNA, and cytokine protein levels, a separate group of rats were used. After behavioral testing on day 11 post-surgery, rats were euthanized by CO2 asphyxiation followed by immediate decapitation. An 18-gauge needle was inserted into the caudal end of the vertebral column and the spinal cord was expelled with ice-cold phosphate buffered saline. The spinal cord was frozen immediately on dry ice and stored at −80 °C until homogenization. The L5 lumbar spinal cord was isolated from the intact frozen cord at the time of mRNA and protein quantification. Total RNA was isolated from the L5 lumbar spinal cord by the TRIzol extraction method (Invitrozen Corp., Carlsbad, CA, USA).
2.8 Real time RT–PCR
Real-time quantitative RT–PCR using Taqman methodology (target RNA is reverse transcribed, amplified, detected, and quantified in real time) was performed to analyze GFAP, a marker for astroglia, and Mac-1, a marker for microglia at the mRNA level. The Taqman probes/primers for GFAP, Mac-1 and GAPDH (GAPDH (glyceraldehyde-3-phosphate); a housekeeping gene) were designed based on the published rat cDNA sequences (accession numbers: U59801 for Mac-1, NM_ 017009 for GFAP and NM_017008 for GAPDH). The primer design software Primer Express (Applied Biosystems, CA, USA) was used to design the primers and probes shown in Table 1. The Taqman probe was dually labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein) at the 5′ end and a fluorescence dye quencher, TAMRA (6-carboxytetramethyl-rhodamine) at the 3′ end. The specificity of the PCR primers, was tested under conventional PCR conditions in a thermocycler (Mastercycler Gradient Eppendorf from Brinkmann Instrument Inc., NY, USA), a single band with expected molecular size was observed for GFAP, Mac-1 and GAPDH analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Prior to the reverse transcription reaction, potentially contaminating residual genomic DNA was eliminated by DNAse I treatment of the total RNA, using the DNA-Free Kit (Ambion, TX, USA). RT and real-time PCR reactions were performed using the High Capacity cDNA Archive Kit (Applied Biosystems, CA, USA) and the Platinum Quantitative PCR Supermix-UDG Kit (InVitrogen, CA, USA). The RT reaction was done in a 100 μl total reaction volume containing: 10 μl of 10× RT buffer, 4 μl of 25× dNTPs, 5 μl of Multiscribe reverse transcriptase, 50 U/μl, 21 μl of RNase free water and 10 μg total RNA in a 50 μl volume. The RT reaction was carried out at 25 °C for 10 min, 37 °C for 120 min and 95 °C for 5 min in the Mastercycler Gradient Eppendorf (Brinkmann Instrument Inc.). The real-time PCR was carried out on the iCycler iQ Multicolor Real Time PCR detection system (Bio-Rad, CA, USA). The real-time PCR reactions were carried out in a total reaction volume of 25 μl containing the final concentration of 1.5 U of Platinum Taq DNA polymerase, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 200 μM dGTP, dCTP, and dATP, 400 μM of dUTP and 1 U of UDG (Uracyl DNA glycosylase), 200nM of forward and reverse primers, 200nM of Taqman probe, 5 μl of a 10-fold dilution of cDNA from the RT step.
Summary of real-time PCR primers and Taqman probesa
2.9 RNase protection assay
Assessment of the temporal cytokine mRNA expression in the L5 lumbar spinal cord was performed using a Ribonuclease Protection Assay technique. A MultiProbe RNase protection assay (RPAse) kit was used (PharMingen, San Diego, CA). Total RNA (15 μg) was hybridized to 32P-labeled anti-sense RNA probes transcribed using the rat cytokine-1 (rCK-1) multi-probe template set (including IL-1α/β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-α/β and IFN-γ, L32, GAPDH), resulting in double-stranded target RNA. After RNAse digestion, protected RNA and probe were resolved on a denaturing polyacrylamide gel and visualized by overnight autoradiography. Quantitative image analysis was employed to compare mRNA levels based on band intensities for each cytokine. The intensity of each band was measured using NIH Image software and assigned an arbitrary unit based on the measured intensity levels. Image intensity for the house keeping gene (L32) and background levels were used to normalize cytokine measurements and compared with the relative levels of mRNA. The relative mean level of cytokine mRNA in different groups of rats (saline or morphine treated in sham or L5-spinal nerve-transected rats) was determined, and levels were normalized to normal animals and reported as ratios to normal.
2.10 Protein estimation by ELISA
Quantitative determination of IL-1β, IL-6 and TNF-α protein was performed on the L5 spinal cord harvested on day 11 post surgery. Tissue homogenization was prepared as previously described (Sweitzer et al., 2001b). In brief, weighed sections of L5 spinal cord were homogenized in homogenization buffer consisting of a protease inhibitor (Boehringer Mannheim, Germany) using Power Gen 125 tissue tearer (Fisher Scientific, Suwanee, GA, USA). Samples were spun at 20 000×g for 30 min at 4 °C. Supernatant was aliquoted and stored at −80 °C for future protein quantification. IL-1β, TNF-α (R&D systems, Minneapolis, MN, USA) and IL-6 (Biosource, Camarillo, CA, USA) protein concentrations were determined utilizing the quantitative sandwich enzyme immunoassay according to the manufacturer's protocol. IL-1β, IL-6 and TNF-α protein quantification was determined by comparing samples to the standard curve generated from the respective kits.
2.11 Statistical analysis
Values are expressed as mean±SEM. Comparisons between groups were performed using analysis of variance (ANOVA) for repeated measurements followed by Tukey–Kramer multiple comparisons test using InStat (GraphPad Software Inc., CA, USA). P values less than 0.05 were considered significant. Acute morphine-induced analgesia in mechanical and thermal tests were expressed as maximal possible effect (%MPE) using the formula: %MPE=(WT−CT)/(CO−CT)×100, where WT is withdrawal latency (in seconds) or threshold (in grams) after morphine/saline treatment, CT is latency before morphine/saline treatment and CO is the cut-off value (i.e. 250 g for mechanical test and 15 s for the tail-flick test). The use of MPE takes account of differences in basal latencies between L5 nerve-transected and control rats so that these differences do not bias the quantification of the antinociceptive effect of the administered drug.
3 Results
3.1 Attenuation of thermal and mechanical hyperalgesia by propentofylline
Before L5 spinal nerve transection, all the animals responded to the noxious heat and mechanical stimuli with a response time 4.9±0.7 s in the tail-flick test, and withdrawal pressure 142±18 g in the mechanical paw withdrawal test. Baseline threshold of animals between the groups exhibited comparable baseline thresholds to both noxious thermal (_P_>0.5) and mechanical (_P_>0.5) stimuli. As in our previous study (Raghavendra et al., 2002), L5 spinal nerve transection produced thermal and mechanical hyperalgesia. Intrathecal administration of propentofylline attenuated the development of thermal and mechanical hyperalgesia in L5 spinal nerve-transected rats. An overall (across the entire study period) statistically significant reduction in both thermal (P<0.01) and mechanical (_P_<0.01) hyperalgesia was observed with 1 and 10 μg propentofylline compared with saline-treated (control) animals. The smaller dose of propentofylline (0.1-μg) did not show a significant (_P_>0.5) difference compared to saline treatment (Fig. 1).
Attenuation of nerve injury-induced thermal and mechanical hyperalgesia by propentofylline. Rats received i.t. 0.1, 1, or 10 μg propentofylline (P) or vehicle daily, initiated 1 h prior to L5 spinal nerve transection (L5Tx). Preventive treatment with 1 (L5Tx+P (1)) and 10 μg (L5Tx+P (10)) of propentofylline produced significant decreases in thermal (tail-flick test) and mechanical (paw-pressure test) hyperalgesia. Development of hyperalgesia is reported as the relative decrease in threshold to noxious thermal and mechanical stimuli compared to basal latency recorded prior to surgery. Values are the mean±SEM. *P<0.01 vs. vehicle-treated control (L5Tx) group.
3.2 Propentofylline treatment spared the analgesic actions of acute morphine in nerve -injured rats
In both thermal and mechanical test paradigms, lower doses of morphine selected (1 and 2 mg/kg) produced significant (P<0.05) analgesic effect in the sham-operated rats. Where as in L5 nerve-transected rats, acute administration lower doses of morphine (1 and 2 mg/kg) had no significant effect on the pre-drug threshold latency (_P_>0.5). As shown in Fig. 2, the dose response curve of acute morphine (1–10 mg/kg, i.v.) in nerve-injured rats showed a clear rightward shift in its anti-nociceptive effect, indicating decreased analgesic effects of morphine following peripheral nerve injury. Chronic administration of propentofylline (10 μg, i.t.) to sham-operated rats did not modulate the analgesic actions of acute morphine tested on post-operative day 11 (_P_>0.05). However, propentofylline treatment significantly (P<0.01) restored the analgesic activity of morphine in rats undergoing L5 spinal nerve transection (Fig. 2).
Reversal of decreased analgesic effect of morphine in nerve-injured rats by propentofylline. Sham and nerve-injured rats received daily i.t. injection of propentofylline (P, 10 μg) initiated 1 h prior to surgery. On postoperative day 11, the acute analgesic effect of morphine (1 to 10 mg/kg, i.v.) was evaluated using tail-flick (thermal) and paw-pressure (mechanical) test. Saline-treated nerve-injured rats (L5Tx) showed decreased analgesic efficacy to morphine. Chronic propentofylline treatment (L5Tx+P(10)) significantly reversed the nerve injury-induced decreased analgesic effect of morphine. Values are the mean±SEM. *P<0.01 vs. sham and +P<0.01 vs. L5-spinal nerve-transected (L5Tx) group.
3.3 Propentofylline treatment attenuated L5 spinal nerve transection-induced glial activation
To determine whether propentofylline was associated with an inhibition of nerve injury-induced glial responses at doses that were anti-hyperalgesic, we used qualitative immunocytochemistry and real time RT–PCR. Immunohistochemical analyses revealed a robust increase in anti-OX-42 (marker for microglia) and anti-GFAP immunostaining in the dorsal horn of the L5 lumbar spinal cord after an L5 spinal nerve transection on post-operative day 11. Chronic propentofylline treatment (10 μg, i.t.) decreased the immunoreactive anti-OX-42 and anti-GFAP in L5-spinal nerve-transected rats (Fig. 3).
Representative photomicrographs of the attenuation of spinal nerve transection-induced microglial (upper panel) and astrocytic (lower panel) activation by propentofylline (P; 10 μg) in the dorsal horn of L5 lumbar spinal cord. Chronic propentofylline (L5Tx+P) treatment resulted in decreased microglial (C) and astrocytic (F) activation compared to (B and E) chronic saline-treated nerve-injured rats (L5Tx). Scale bar: 150 μm.
Analysis of transgene expression by real time RT–PCR showed the constitutive expression of Mac-1 and GFAP at L5 lumbar spinal cord of the non-operative (normal) rats. Following L5 spinal nerve transection (on postoperative day 11), expression of Mac-1 and GFAP were significantly elevated over normal (P<0.01) and control (_P_<0.01) groups of animals. Chronic propentofylline treatment (10 μg, i.t.) to L5 spinal nerve-transected rats significantly (_P_<0.01) reduced the expression of mRNA for _Mac-1_ and _GFAP_ compared to vehicle-treated neuropathic rats (Fig. 4). However, chronic propentofylline treatment had no significant (_P_>0.05) effect on levels of mRNA for Mac-1 and GFAP in normal and sham-operated rats (data not shown).
Relative expression of mRNA for Mac-1 and GFAP in propentofylline treated L5 spinal nerve-transected rats. Chronic treatment with propentofylline (P; 10 μg) to nerve-injured rats (L5Tx+P) significantly decreased the expression of mRNA for Mac-1 and GFAP compared to saline-treated nerve-injured rats (L5Tx). Values are mean±SEM (_n_=4). *P<0.01 vs. sham, + P<0.01 vs. L5Tx rats (_n_=4/group).
3.4 Propentofylline treatment inhibits L5 spinal nerve transection-induced upregulation of proinflammatory cytokines in the L5 lumbar spinal cord
Constitutive expression of mRNA for IL-α/β, TNF-α/β and IL-6 was observed in L5 lumbar spinal cord of control and sham-operated rats. Following an L5 spinal nerve transection (on post operative day 11), a significant (P<0.01) increase in mRNA levels of IL-α/β, TNF-α/β and IL-6 was observed as compared to sham-operated animals. Chronic administration of propentofylline (10 μg, i.t.) to L5 spinal nerve-transected rats significantly (P<0.01) attenuated the upregulation of mRNA for IL-1α/β, TNF-α/β and IL-6 compared to saline-treated rats. Unlike the constitutive expression of specific proinflammatory cytokines, mRNA for the anti-inflammatory cytokine, IL-10 was not observed in control or sham-operated rats. However, following L5 spinal nerve transection, a significant (P<0.01) increase in mRNA for IL-10 was observed as compared to normal and sham-operated rats. Chronic administration of propentofylline further enhanced (P<0.01) nerve injury-associated IL-10 mRNA expression at L5 lumbar spinal cord (Fig. 5 and Table 2). L5 spinal nerve transection significantly (P<0.01) increased IL-1β, IL-6 and TNF-α protein levels as compared to sham-operated rats, which was significantly (P<0.01 vs. vehicle-treated neuropathic rats) attenuated by chronic administration of propentofylline (10 μg, i.t.) (Table 3).
Representative RNase protection assay demonstrating cytokine mRNA expression in L5 lumbar spinal cord of control (C), sham (S), saline-treated L5 spinal nerve-transected (L5Tx) and propentofylline (P; 10 μg)-treated L5Tx rats (L5Tx+P) (_n_=4/group).
Effect of chronic administration of propentofylline on spinal cytokine mRNA in L5 spinal nerve transected and sham operated rats
Effect of chronic administration of propentofylline on spinal levels of proinflammatory cytokines L5-spinal nerve transected, sham surgery and control rats
4 Discussion
Propentofylline, a methylxanthine derivative exhibits neuroprotective effects through multiple mechanisms. These include: an inhibition of glutamate release (Andine et al., 1990; Miyashita et al., 1992), an increase in nerve growth factor secretion (Shinoda et al., 1990) and attenuation of glial activation (Schubert et al., 2000). The specific mechanism by which propentofylline exhibits such diverse effects is not understood due to its multiple mechanisms of actions, which include: non-specific inhibition of phosphodiesterase enzymes (Meskini et al., 1994; Schubert et al., 1997) and its ability to inhibit adenosine re-uptake (Parkinson et al., 1993). Both these molecular mechanisms exert neuroprotective effects. In the present study, similar to the anti-allodynic properties that were previously described (Sweitzer et al., 2001c), we observed prolonged anti-hyperalgesic properties of propentofylline 15 h after administration. This supports a mechanism of action that includes modulation of intracellular second messenger signaling since the half-life of propentofylline and its active metabolite is about 1 h (Sweitzer et al., 2001c).
Similar to earlier observations of anti-allodynic effects (Sweitzer et al., 2001c), the anti-hyperalgesic activity of propentofylline was also associated with its ability to suppress spinal glial activation induced by peripheral nerve transection. Propentofylline attenuated nerve injury-induced up-regulation of mRNA for Mac-1 and GFAP, markers for activated microglia and astrocytes, respectively, and decreased immunostaining for OX-42 and GFAP in the dorsal horn of the lumbar spinal cord. These results suggest propentofylline acts both on microglia and astrocytes to inhibit gliosis associated with peripheral nerve injury. Activated glia releases various neuroactive substances like reactive oxygen species, nitric oxide, prostaglandins, excitatory amino acids and cytokines (Watkins et al., 2001a,b). Excessive synaptic accumulation of these products is associated with neuronal hyperactivity, which is an important element in the development and maintenance of persistent pain states (DeLeo and Yezierski, 2001; Watkins et al., 2001a,b). It is important to note that propentofylline did not ameliorate the glial activation but decreased the expression of relevant surface markers (see Fig. 4). This underscores the mechanism of action of propentofylline as a glial modulating agent not a glial suppressor. This has obvious implications since glia also are pivotal players in healing, regeneration and synaptic homeostasis (Kreutzberg, 1996; Streit, 2002).
The release of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α from activated glia in the CNS may be responsible for the development of central sensitization associated with peripheral nerve injury or inflammation (DeLeo and Yezierski, 2001). In the present study, apart from the modulation of glial activation, propentofylline treatment also down regulated the mRNA and protein expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in the lumbar spinal cord of L5 spinal nerve-transected rats. This suggests anti-allodynic and anti-hyperalgesic actions of propentofylline are also attributed to its ability to modulate central proinflammatory immune responses.
The mechanism of suppression of proinflammatory cytokines synthesis and release by propentofylline in nerve-injured rats could be due to its inhibitory action on phosphodiesterase enzymes and subsequent augmentation of cAMP signaling. This is supported by work in which administration of propentofylline mimicked the dibutyryl-cAMP action in inhibiting LPS-induced release of IL-1β, TNF-α and oxygen radical from cultured microglial cells (Si et al., 1996, 1998). In addition, similar to the cAMP signaling-induced upregulation of anti-inflammatory IL-10 (Platzer et al., 1999), propentofylline treatment also enhanced mRNA for IL-10 in nerve-injured rats. This highlights that addition to the suppression of proinflammatory cytokine, propentofylline also has the ability to enhance anti-inflammatory cytokines. Of particular relevance to this, it has been previously shown that in a rat model of mononeuropathy application of IL-10 at the site of injury decreased the development of hyperalgesia (Wagner et al., 1998).
As an atypical methylxanthine, propentofylline also functions as an adenosine reuptake inhibitor (Parkinson et al., 1993). This is potentially important because adenosine has been proposed to have a role in neuropathic pain. A decreased level of adenosine in the CSF of patients suffering from neuropathic pain has been reported (Guieu et al., 1996). Reduced efficacy of spinal morphine in nerve injury-induced allodynia and hyperalgesia reflect a disruption in spinal opioid-adenosine mechanism (Lavand'homme and Eisenach, 1999; Sandner-Kiesling et al., 2001). Intrathecal administration of adenosine not only reversed the allodynia and hyperalgesia in nerve-injured rats, but also restored morphine analgesia in these animals (Lavand'homme and Eisenach, 1999; Sandner-Kiesling et al., 2001). Adenosine receptor agonists (for A1 and A2a) have been found to inhibit microglial proliferation (Si et al., 1996), and exhibit antiallodynic property in both inflammatory and neuropathic pain models (Sawynok, 1998). Signaling through A2a receptor also strengthens cAMP-dependent signaling by enhancement of adenylate cyclase.
Activation of glia and elevation of spinal proinflammatory cytokines not only induce central sensitization and behavioral hypersensitivity, but also modulate the antinociceptive properties of morphine (Raghavendra et al., 2002). The proinflammatory cytokine, IL-1β is involved in the antianalgesic activity of diverse agents such as dynorphin against morphine analgesia (Laughlin et al., 2000; Rady and Fujimoto, 2001). Indeed, disrupting glial activation or inhibition of spinal proinflammatory immune responses attenuates morphine tolerance, and restores the acute antinociceptive effect of morphine in sham and neuropathic rats (Song and Zhao, 2001; Raghavendra et al., 2002). In the present study, chronic propentofylline treatment had no effect on acute morphine-induced analgesia in normal or sham-operated rats. However, it significantly restored the analgesic activity of morphine in neuropathic rats, which is associated with its ability to inhibit glial activation and spinal proinflammatory cytokine levels. These findings suggest the importance of proinflammatory cytokines in decreasing morphine analgesia in neuropathic pain conditions. Similar to propentofylline, pentoxifylline, another xanthine derivative showed attenuation of post injury-induced hyperalgesia and enhanced morphine analgesia in rats (Wordliczek et al., 2000). Also, pentoxifylline treatment decreased the postoperative pain in patients, which is associated with its ability to inhibit proinflammatory immune responses (Wordliczek et al., 2000).
This study along with our earlier observations demonstrates that propentofylline is a novel anti-allodynic and anti-hyperalgesic agent that is capable of modulating spinal glial activation and proinflammatory immune responses associated with peripheral nerve injury. Its mechanism may be distinct from other pharmacological and surgical interventions that have demonstrated significant efficacy in reducing or ameliorating hypersensitivity after nerve injury (Ossipov et al., 2000; Wang et al., 2001; Burgess et al., 2002). These studies and others certainly highlight the multiple mechanisms that give rise to behavioral hypersensitivity following nerve injury.
The positive safety profile of long-term propentofylline treatment (Mielke et al., 1998) and its ability to spare morphine analgesia may have widespread clinical implications. Propentofylline may increase the options for effective neuropathic pain treatment or reduce opioid dose escalation which is often associated with unwanted side effects and the development of tolerance. In summary, these data support a role of immunocompetent glia and spinal neuroimmunologic processes in both the generation and maintenance of persistent pain and modulation of morphine analgesia following traumatic nerve injury.
Acknowledgements
The authors would like to thank Tracy Wynkoop for editorial assistance, Dr. William F. Hickey for antibodies and glial expertise; and the following for grant support: National Institute of Drug Abuse grant DA11276 (J.A.D.).
References
Andine P, Rudolphi KA, Fredholm BB, Hagberg H. Effect of propentofylline (HWA 285) on extracellular purines and excitatory amino acids in CA1 of rat hippocampus during transient ischaemia. Br J Pharmacol. 1990;100:814-818.
Bao L, Zhu Y, Elhassan AM, Qinyang W, Xiao B, Zhu J, Lindgren JU. Adjuvant-induced arthritis: IL-1β, IL-6 and TNF-α are up-regulated in the spinal cord. Neuroreport. 2001;12:3905-3908.
Bethea JR, Castro M, Keane RW, Lee TT, Dietrich WD, Yezierski RP. Traumatic spinal cord injury induces nuclear factor-kappaB activation. J Neurosci. 1998;18:3251-3260.
Bian D, Ossipov MH, Ibrahim M, Raffa RB, Tallarida RJ, Malan TP Jr, Lai J, Porreca F. Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphine antiserum. Brain Res. 1999;831:55-63.
Burgess SE, Gardell LR, Ossipov MH, Malan TP Jr, Vanderah TW, Lai J, Porreca F. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J Neurosci. 2002;22:5129-5136.
Colburn RW, Rickman AJ, DeLeo JA. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp Neurol. 1999;157:289-304.
Covino BG, Dubner R, Gybels J, Losterlitz HW, Liebeskind JC, Sternbach RA, Vylicky L, Yamamura H, Zimmerman M. Ethical standards for investigations of experimental pain in animals. Pain. 1980;9:141-143.
DeLeo JA, Colburn RW., 1999. Proinflammatory cytokines and glial cells: their role in neuropathic pain. In: Watkins LR, Maier SF, editors., Cytokines and pain. Birkhauser, Basel, pp. 159-181.
DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain. 2001;90:1-6.
Guieu R, Peragut JC, Roussel P, Hassani H, Sampieri F, Bechis G, Gola R, Rochat H. Adenosine and neuropathic pain. Pain. 1996;68:271-274.
Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 1996;19:312-318.
Laughlin TM, Bethea JR, Yezierski RP, Wilcox GL. Cytokine involvement in dynorphin-induced allodynia. Pain. 2000;84:159-167.
Lavand'homme PM, Eisenach JC. Exogenous and endogenous adenosine enhance the spinal antiallodynic effects of morphine in a rat model of neuropathic pain. Pain. 1999;80:31-36.
Martin TJ, Eisenach JC. Pharmacology of opioid nonopioid analgesics in chronic pain states. J Pharmacol Exp Ther. 2001;238:352-359.
Meller ST, Dyskstra C, Grzybycki D, Murphy S, Gebhart GF. The possible role of glia in nociceptive processing and hyperalgesia in the spinal cord of the rat. Neuropharmacology. 1994;33:1471-1478.
Meskini N, Nemoz G, Okyayuz-Baklouti I, Lagard M, Prigent AF. Phosphodiesterase inhibitory profile of some related xanthine derivatives pharmacologically active on the peripheral microcirculation. Biochem Pharmacol. 1994;47:781-788.
Mielke R, Moller HJ, Erkinjuntti T, Rosenkranz B, Rother M, Kittner B. Propentofylline in the treatment of vascular dementia and Alzheimer type dementia: overview of phase 1 and phase II clinical trials. Alzheimer Dis Assoc Disord. 1998;12:S29-S35.
Milligan ED, O'Connor KA, Nguyen KT, Armstrong CB, Twining C, Gaykema RPA, Holguin A, Martin D, Maier SF, Watkins LR. Intrathecal HIV-1 envelope glycoprotein gp 120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines. J Neurosci. 2001;21:2808-2819.
Miyashita K, Nakajima T, Ishikawa A, Miyatake T. An adenosine uptake blocker, propentofylline, reduces glutamate release in gerbil hippocampus following transient forebrain ischemia. Neurochem Res. 1992;17:147-150.
Ossipov MH, Lai J, Malan TP Jr, Porreca F. Spinal and supraspinal mechanisms of neuropathic pain. Ann NY Acad Sci. 2000;909:12-24.
Parkinson FE, Paterson ARP, Young JD, Cass CE. Inhibitory effects of propentofylline on [3H]adenosine influx. Biochem Pharmacol. 1993;46:891-896.
Platzer C, Fritsch E, Elsner T, Lehmann MH, Volk HD, Prosch S. Cyclic adenosine-monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells. Eur J Immunol. 1999;29:3098-3104.
Rady JJ, Fujimoto JM. Confluence of antianalgesic action of diverse agents through brain interleukin1α in mice. J Pharmacol Exp Ther. 2001;299:659-665.
Raghavendra V, Rutkowski MD, DeLeo JA. The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham operated rats. J Neurosci. 2002;22:9980-9989.
Sandner-Kiesling A, Li X, Eisenach JC. Morphine-induced spinal release of adenosine is reduced in neuropathic rats. Anesthesiology. 2001;95:1455-1459.
Sawynok J. Adenosine receptor activation and nociception. Eur J Pharmacol. 1998;347:1-11.
Schubert P, Ogata T, Rudolphi K, Marchini C, McRae A, Ferroni S. Support of homeostatic glial cell signaling: a novel therapeutic approach by propentofylline. Ann NY Acad Sci. 1997;826:337-347.
Schubert P, Morino T, Miyazaki H, Ogata T, Nakamura Y, Marchini C, Ferroni S. Cascading glia reactions: a common pathomechanism and its differentiated control by cyclic nucleotide signaling. Ann NY Acad Sci. 2000;903:24-33.
Shinoda I, Furukawa Y, Furukawa S. Stimulation of nerve growth factor synthesis/secretion by propentofylline in cultured mouse astroglial cells. Biochem Pharmacol. 1990;39:1813-1816.
Si Q, Nakamura Y, Schubert P, Rudolphi K, Kataoka K. Adenosine and propentofylline inhibit the proliferation of cultured microglial cells. Exp Neurol. 1996;137:345-349.
Si Q, Nakamura Y, Ogata T, Kataoka K, Schubert P. Differential regulation of microglial activation by propentofylline via cAMP signaling. Brain Res. 1998;812:97-104.
Song P, Zhao Z-Q. The involvement of glial cells in the development of morphine tolerance. Neurosci Res. 2001;39:281-286.
Stein C, Gramsch C, Herz A. Intrinsic mechanisms of antinociception in inflammation: local opioid receptors and beta-endorphin. J Neurosci. 1990;10:1292-1298.
Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia. 2002;40:133-139.
Sweitzer SM, Arruda JL, DeLeo JA., 2001. The cytokine challenge: methods for the detection of central cytokines in rodent models of persistent pain. In: Kruger L, editor., Methods in pain research. CRC Press, New York, pp. 109-132.
Sweitzer SM, Martin D, DeLeo JA. Intrathecal interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibits an anti-allodynic action in a rat model of neuropathic pain. Neuroscience. 2001;103:529-539.
Sweitzer SM, Schubert P, DeLeo JA. Propentofylline, a glial modulating agent, exhibits anti-allodynic properties in a rat model of neuropathic pain. J Pharmacol Exp Ther. 2001;297:1210-1217.
Wagner R, Janjigian M, Myers RR. Anti-inflammatory interleukin-10 therapy in CCI neuropathy decreases thermal hyperalgesia, macrophage recruitment, and endoneurial TNF-alpha expression. Pain. 1998;74:35-42.
Wang Z, Gardell LR, Ossipov MH, Vanderah TW, Brennan MB, Hochgeschwender U, Hruby VJ, Malan TP Jr, Lai J, Porreca F. Pronociceptive actions of dynorphin maintain chronic neuropathic pain. J Neurosci. 2001;21:1779-1786.
Watkins LR, Martin D, Ulrich P, Tracey KJ, Maier SF. Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat. Pain. 1997;71:225-235.
Watkins LR, Wiertelak BP, Goehier LB, Smith KP, Martin D, Maier SF. Characterization of cytokine-induced hyperalgesia. Brain Res. 1994;654:15-26.
Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain. Trends Neurosci. 2001;24:450-455.
Watkins LR, Milligan ED, Maier SF. Spinal cord glia: new players in pain. Pain. 2001;93:201-205.
Wordliczek J, Szczepanik AM, Banach M, Turchan J, Zembala M, Siedlar M, Przewlocki R, Serednicki W, Przewocka B. The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients. Life Sci. 2000;66:1155-1164.
Zimmermann M. Pathobiology of neuropathic pain. Eur J Pharmacol. 2001;429:23-37.
Keywords:
Propentofylline; Peripheral nerve injury; Proinflammatory cytokine; Spinal glia; Rat
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