Minocycline and pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain

European Journal of Pharmacology 560 (2007) 142 – 149 www.elsevier.com/locate/ejphar Minocycline and pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain Joanna Mika, Maria Osikowicz, Wioletta Makuch, Barbara Przewlocka ⁎ Department of Pain Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Cracow, Poland Received 28 July 2006; received in revised form 21 December 2006; accepted 8 January 2007 Available online 19 January 2007 Abstract Recent research has shown that microglial cells which are strongly activated in neuropathy can influence development of allodynia and hyperalgesia. Here we demonstrated that preemptive and repeated i.p., administration (16 h and 1 h before injury and then after nerve ligation twice daily for 7 days) of minocycline (15; 30; 50 mg/kg), a potent inhibitor of microglial activation, significantly attenuated the allodynia (von Frey test) and hyperalgesia (cold plate test) measured on day 3, 5, 7 after chronic constriction injury (CCI) in rats. Moreover, the 40% improvement of motor function was observed. In mice, i.p., administration of minocycline (30 mg/kg) or pentoxifylline (20 mg/kg) according to the same schedule also significantly decreased allodynia and hyperalgesia on day 7 after CCI. Antiallodynic and antihyperalgesic effect of morphine (10 mg/kg; i.p.) was significantly potentiated in groups preemptively and repeatedly injected with minocycline (von Frey test, 18 g versus 22 g; cold plate test, 13 s versus 20 s in rats and 1.2 g versus 2.2 g; 7.5 s versus 10 s in mice; respectively) or pentoxifylline (1.3 g versus 3 g; 7.6 s versus 15 s in mice; respectively). Antiallodynic and antihyperalgesic effect of morphine (30 μg; i.t.) given by lumbar puncture in mice was also significantly potentiated in minocycline-treated group (1.2 g versus 2.2 g; 7.5 s versus 11 s; respectively). These findings indicate that preemptive and repeated administration of glial inhibitors suppresses development of allodynia and hyperalgesia and potentiates effects of morphine in rat and mouse models of neuropathic pain. © 2007 Elsevier B.V. All rights reserved. Keywords: Neuropathic pain; Allodynia; Hyperalgesia; Morphine; Microglia; Glial inhibitors 1. Introduction For years, microglial cells were considered to have only a supportive and nutritive function in the central nervous system. However, recent lines of evidence have strongly suggested that they may play a more active role in synaptic functions than previously thought. A striking feature of microglia cells reactivity is their ability to synthesize and secrete a large number of substances including growth factors, cytokines, complement factors, lipid mediators, extracellular matrix components, enzymes, free radicals, neurotoxins, nitric oxide and prostaglandins (Minghetti and Levi, 1998). Although low levels of these inflammatory mediators can play a vital role in the regeneration and repair of damaged tissue, high levels of these products have been reported to contribute to neuronal degeneration (Minghetti and Levi, ⁎ Corresponding author. Tel.: +48 12 6623398; fax: +48 12 6374500. E-mail address: przebar@if-pan.krakow.pl (B. Przewlocka). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.01.013 1998). A number of studies have shown remarkable changes in microglial morphology and functions under pathological conditions, such as disease or injury (Eriksson et al., 1993; Okimura et al., 1996; Quattrini et al., 1996; Roper et al., 1997; Franke et al., 1999). It seems that microglial activation after peripheral nerve injury can be a causal factor particularly important in the initiation of neuropathic pain (Eriksson et al., 1993; Kreutzberg, 1996; Coyle, 1998; Marchand et al., 2005; Ledeboer et al., 2005), because microglia release proinflammatory cytokines which were shown to be common mediators of allodynia and hyperalgesia (Eriksson et al., 1993; Wagner et al., 1995; Liu et al., 1995; Boddeke, 2001; Raghavendra et al., 2004; Watkins and Maier, 2003). Moreover, uncontrolled activation of microglial cells after injury can lead to altered activity of opioid systems or opioidspecific signaling (Speth et al., 2002; Przewlocki and Przewlocka, 2005). The impairment of opioidergic transmission can be the reason of reduction of antinociceptive potency of morphine after nerve injury as consequence of decreased number of presynaptic J. Mika et al. / European Journal of Pharmacology 560 (2007) 142–149 opioid receptors induced by loss of neurons (Ossipov et al., 1995; Porreca et al., 1998; Speth et al., 2002; Przewlocki and Przewlocka, 2005). Minocycline, a semisynthetic second-generation tetracycline with adequate penetration into the brain and cerebrospinal fluid (Aronson, 1980; Wu et al., 2002; Colovic and Caccia, 2003), has emerged as a potent inhibitor of microglial activation and proliferation, devoid of any known direct action on astrocytes or neurons (Amin et al., 1996; Tikka and Koistinaho, 2001; Tikka et al., 2001). Recently, minocycline has been shown to be an effective neuroprotective agent in animal models of spinal cord injury (Stirling et al., 2004), Parkinson's disease (Wu et al., 2002; Blum et al., 2004), multiple sclerosis (Brundula et al., 2002) and focal ischemia (Yrjanheikki et al., 1998, 1999). Beneficial effects of minocycline are also associated with reduction of inducible nitric oxide synthase and cyclooxygenase-2 expression, decrease in cytokine and prostaglandin release and a decrease in induction of IL-1beta-converting enzyme in microglia (Yrjanheikki et al., 1998, 1999). Moreover, it was demonstrated that the administration of minocycline either systemically or intrathecally attenuated hyperalgesia in various rat models of neuropathy, and these effects depended on inhibition of spinal glial activation and consequently lowered expression of proinflammatory cytokines (Raghavendra et al., 2003; Ledeboer et al., 2005; Li et al., 2005). Recent reports revealed that preemptive systemic minocycline administration blocked the development of neuropathic pain states in rats in L5 nerve transection model, but did not reduce pain that was already established (Raghavendra et al., 2003). Ledeboer et al. (2005) suggested that microglia cells play a crucial role in initiating, rather than maintaining, enhanced pain responses. Such influence was previously described for pentoxifylline; a non-specific cytokine inhibitor and inhibitor of phosphodiesterase (Sweitzer et al., 2001; Raghavendra et al., 2003, 2004). This drug inhibited the synthesis of TNF-alpha, IL-1, IL-6, and IL-8 (Neuner et al., 1994; Lundblad et al., 1995). A few studies demonstrated that pentoxifylline diminished the development of formalin-induced pain behavior in rats (Dorazil-Dudzik et al., 2004) and post-operative pain in patients (Wordliczek et al., 2000; Szczepanik et al., 2004). Furthermore, preemptive administration of pentoxifylline influenced morphine intake in postoperative period in patient-controlled analgesia (Wordliczek et al., 2000). Recent behavioral studies have shown restoration of the analgesic activity of morphine by propentofylline treatment in neuropathic pain (Sweitzer et al., 2001; Raghavendra et al., 2002, 2004) and spinal infusion of fluorocitrate (glial metabolic inhibitor) after intraperitoneal injection of a bacterial endotoxin, lipopolysaccharide (Johnston and Westbrook, 2005). Therefore, we investigated how two glial inhibitors administered i.p. preemptively and repeatedly influence allodynia, hyperalgesia and improve recovery of the motor function in rat and mice after sciatic nerve injury. Moreover, it was interesting to examine a possible influence of preemptive and repeated minocycline or pentoxifylline administration on effectiveness of morphine (10 mg/kg i.p. and 30 μg; i.t.) in rat and mouse models of neuropathic pain. 143 2. Materials and methods 2.1. Animals Male Wistar rats (200–350 g) and Albino-Swiss mice (20–25 g) were housed in cages lined with sawdust, under standard 12/12 h light/dark cycle (lights on at 08:00 h) with food and water ad lib. All experiments were performed according to the recommendations of IASP (Zimmermann, 1983), the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the local Bioethics Committee (Cracow, Poland). 2.2. Chronic constriction injury Chronic constriction injury (CCI) was produced according to Bennett and Xie (1988). The right sciatic nerve was exposed under sodium pentobarbital anesthesia (60 mg/kg; i.p.). Four ligatures (4/0 silk) for rats and three for mice were made around the nerve distal to the sciatic notch with 1 mm spacing, until a brief twitch in the respective hind limb was observed. After CCI all animals developed allodynia and hyperalgesia. 2.3. Intrathecal administration The rats were chronically implanted with intrathecal (i.t.) catheters according to Yaksh and Rudy (1976) under pentobarbital anesthesia (60 mg/kg; i.p.). The catheter (PE 10, INTRAMEDIC, Clay Adams, Becton Dickinson and Company, Rutherford, NJ, USA) sterilized by immersion in 70% ethanol, and flushed with sterile water prior to insertion was carefully introduced through atlanto-occipital membrane to the subarachnoid space at the rostral level of the spinal cord lumbar enlargement (L4–L6). The CCI to the sciatic nerve was made 5 days after catheter implantation. Intrathecal injections were carried out in rats 12 days after catheter implantation and on day 7 after CCI. In mice, intrathecal administrations were carried out by lumbar puncture according to Hylden and Wilcox (1980) 7 days after CCI. 2.4. Drug administration Chemicals and their sources were as follows: minocycline hydrochloride (Sigma, USA); pentoxifylline (Polfilin, Polfarma, Poland) and morphine hydrochloride (Polfa Kutno, Poland). All drugs were dissolved in sterile water. Minocycline in rats (15, 30, 50 mg/kg; i.p.) and mice (15, 30 mg/kg; i.p.) as well as pentoxifylline in mice (15, 20 mg/kg; i.p.) were administered preemptively: 16 h and 1 h before CCI and then twice daily for 7 days. The control groups received vehicle according to the same schedule. The behavioral tests were conducted on day 3, 5 or 7 after CCI 60 min (rats) or 30 min (mice) after minocycline or pentoxifylline i.p. administration. On day 7 after the tests, morphine (10 mg/kg; i.p. or 30 μg; i.t.) was injected and 30 min later the same behavioral tests were repeated. 144 J. Mika et al. / European Journal of Pharmacology 560 (2007) 142–149 2.5. Behavioral tests 2.5.1. Tactile allodynia (von Frey test) Allodynia was measured in CCI rats by the use of automatic von Frey apparatus (Dynamic Plantar Anesthesiometer Cat. No. 37400, Ugo Basile Italy). Animals were placed in plastic cages with wire net floor. The von Frey filament was applied to the midplantar surface of the hind paw and the measurements were taken automatically, as described previously by Chaplan et al. (1994). In rats, the strength of the von Frey stimuli ranged from 0.5 to 26 g. Allodynia in mice was measured by applying calibrated nylon von Frey monofilaments (Stoelting, USA) at increasing strength (0.5–6 g) sequentially to the plantar surface of the hind paw of each mice until the hind paw was withdrawn (Sommer and Schafers, 1998). 2.5.2. Cold hyperalgesia (cold plate test) Thermal hyperalgesia was assessed using the cold plate test (Cold/Hot Plate Analgesia Meter No. 05044 Columbus Instruments, USA). The temperature of the cold plate was kept at 5 °C for rats and at 2 °C for mice. The animals were placed on the cold plate and the time until lifting of the hind paw was recorded. The cut-off latency was 30 s. 2.5.3. Warm hyperalgesia (paw withdrawal test) Thermal hyperalgesia in rats was tested for paw withdrawal latency to thermal stimuli using an Analgesia Meter (Landing, NJ). Rats were placed into the individual plastic cages with the glass floor 5 min before the experiment. Noxious thermal stimulus was focused through glass onto the plantar surface of a hind paw until the animal lifted the paw away. The cut-off latency was 20 s. 2.5.4. Motor functions (walking pattern test) The return of motor function in rats was monitored by analysis of the free walking pattern according to the De Medinacelli et al. (1982) with De Koning et al. (1986) modifications. This standardized method yields a reproducible measure in motor deficit of the involved paw. The functional index (FI) of paw motor function is expressed in % as the difference between the ipsi- and contralateral paw. After injury, the functional index increases to 77.5% ± 2.3, indicating the effectiveness of the lesion. During regeneration, the return of function in the paw gradually decreases the functional index toward 0%. One week before experiments the animals were trained to walk over the strips of paper covering the bottom of a 50 cm long, 6.4 cm wide, upward inclining (10°) corridor ending with a dark box. The rats' hind limbs were dipped in ink and their footprints were recorded in the corridor on the white paper. The results were analyzed using a computer program. Motor functions were recorded on day 3, 5, 7 after sciatic nerve injury. 2.6. Data analysis The behavioral data are presented as the mean ± S.E.M. of 8–16 animals per group. The results of the experiments were Fig. 1. Influence of preemptive and repeated administration of minocycline on the development of allodynia, hyperalgesia and motor function after CCI in rats. Effect of minocycline (15, 30 or 50 mg/kg; i.p.; 16 h and 1 h before CCI and then for 7 days twice daily) on the development of mechanical allodynia (A; von Frey test), hyperalgesia (B; cold plate test, C; paw withdrawal test) and on the motor function (D; walking pattern test) 3, 5, 7 days after CCI in rats. The data are presented as the mean ± S.E.M (10–12 rat per group). Allodynia and hyperalgesia were assessed 60 min after drug administration. Inter-group differences were analyzed by ANOVA Bonferroni's Multiple Comparison Test; ⁎ P b 0.05; ⁎⁎ P b 0.01; ⁎⁎⁎P b 0.001 indicates a significant difference compared with control (vehicle-treated CCI rats). J. Mika et al. / European Journal of Pharmacology 560 (2007) 142–149 145 evaluated by one-way analysis of variance (ANOVA). The differences between groups were further analyzed with Bonferroni post-hoc test. ⁎ P b 0.05; ⁎⁎ P b 0.01; ⁎⁎⁎ P b 0.001 indicate a significant difference vs. control (vehicle-treated CCI mice or vehicle-treated CCI mice which received a single dose of morphine). 3. Results 3.1. The effect of preemptive and repeated minocycline or pentoxifylline treatment on the CCI-induced changes in rats and mice 3.1.1. The influence of glial inhibitors on CCI-induced development of allodynia, hyperalgesia and motor function in rats All vehicle-treated CCI rats exhibited strong allodynia starting from day 3 after injury. On day 7 after CCI, allodynia reached the value of 11.71 g ± 0.7 according to the von Frey test compared to 25.3 g ± 0.2 in uninjured rats. Other behavioral tests revealed that rats developed a potent thermal hyperalgesia in the cold plate test (5.83 s ± 0.5 vs. 30 s ± 0 in control) and paw withdrawal test (4.24 s ± 0.26 vs. 8.1 s ± 0.5 in control). Preemptive treatment with minocycline (15, 30 or 50 mg/kg; i.p.) at 16 h and 1 h before CCI and then 7-day treatment twice daily attenuated the development of neuropathic pain symptoms in rats measured on 3, 5 and 7 days after CCI (Fig. 1). The low dose (15 mg/kg, i.p.) of minocycline was effective only in alleviating mechanical allodynia, but not in lessening thermal hyperalgesia. However, higher doses (30 and 50 mg/kg; i.p.) attenuated allodynia and hyperalgesia to similar extent. The influence was significant on day 3, 5 and 7 after CCI (Fig. 1). Fig. 2. Influence of preemptive and repeated administration of minocycline or pentoxifylline on development of allodynia and hyperalgesia after CCI in mice. Effect of minocycline (MC; 15 and 30 mg/kg; i.p.) and pentoxifylline (PF; 15 and 20 mg/kg; i.p.) 16 h and 1 h before CCI and then for 7 days twice daily on the development of mechanical allodynia (A; von Frey test) and hyperalgesia (B; cold plate test) after CCI in mice. The data are presented as the mean ± S.E.M. (8–12 mice per group). Allodynia and hyperalgesia were assessed 30 min after drug administration. Inter-group differences were analyzed by ANOVA Bonferroni's Multiple Comparison Test; ⁎⁎ P b 0.01; ⁎⁎⁎ P b 0.001 indicates a significant difference compared with control (vehicle-treated CCI mice). Fig. 3. The influence of preemptive and repeated administration of minocycline or pentoxifylline on the effect of a single intraperitoneal dose of morphine (10 mg/kg; i.p.) on day 7 after CCI in rats and mice. In rats, von Frey (A) and cold plate (B) tests were conducted 60 min after the last dose of minocycline (MC; 30 mg/kg; i.p.; 16 h and 1 h before CCI and then for 7 days twice daily). Morphine (10 mg/kg; i.p.) was injected following the tests and the same tests were repeated 30 min thereafter. In mice, von Frey (C; E) and cold plate (D; F) tests were conducted 30 min after the last dose of minocycline (MC; 30 mg/kg; i.p.; 16 h and 1 h before CCI and then 7 days twice daily) or pentoxifylline (PF; 20 mg/kg; i.p.; 16 h and 1 h before CCI and then 7 days twice daily). Morphine (10 mg/kg; i.p.) was injected following the tests and the same tests were repeated 30 min thereafter. The data are presented as the mean ± S.E.M. (8–16 animals per group). Inter-group differences were analyzed by ANOVA Bonferroni's Multiple Comparison Test; ⁎ P b 0.05; ⁎⁎ P b 0.01; ⁎⁎⁎ P b 0.001 indicates a significant difference compared with control (vehicle-treated CCI animals or vehicle-treated CCI animals which received a single dose of morphine). Calculation of the functional index from the preoperative footprints indicated a normal paw function (approximately 0% of functional impairment). The sciatic nerve injury impaired 146 J. Mika et al. / European Journal of Pharmacology 560 (2007) 142–149 motor function on day 3 after CCI, and the functional index was approximately 77.5% ± 2.3 for the control group (vehicle-treated CCI rats). Minocycline-treated rats (15, 30 or 50 mg/kg) had better time course of recovery (Fig. 1D). The significant effects of different doses of minocyclinetreated vs. vehicle-treated rats were observed already on day 3. The most beneficial effect was observed at the highest dose of minocycline (50 mg/kg; i.p.), for example functional index on day 7 was 41.6 % ± 5.2 in minocycline-treated vs. 66.7 % ± 2.7 in vehicle-treated rats. 3.1.2. The influence of glial inhibitors on CCI-induced development of allodynia and hyperalgesia in mice In mice, preemptive treatment (16 h and 1 h before CCI) followed by 7-day administration of minocycline (twice daily; 15 and 30 mg/kg; i.p.) and pentoxifylline (twice daily; 15 and 20 mg/kg; i.p.) dose-dependently attenuated the development of mechanical allodynia (Fig. 2A) and thermal hyperalgesia (Fig. 2B) as measured on day 7 after CCI. 3.2. The effect of preemptive and repeated minocycline or pentoxifylline treatment on the effects of morphine on the CCIinduced changes in rats and mice 3.2.1. The influence of glial inhibitors on morphine intraperitoneal administration in rats and mice In CCI rats, the dose of morphine (10 mg/kg; i.p.) only slightly diminished allodynia (von Frey test) and hyperalgesia (cold plate test) (Fig. 3). Preemptive treatment (16 h and 1 h before CCI) followed by 7-day administration of minocycline (twice daily; 30 mg/kg; i.p.) improved the response to morphine (10 mg/kg; i.p.). The effect of morphine in minocycline-treated CCI rats was significantly higher in both tests in comparison with vehicle-treated CCI rats injected with morphine (Fig. 3A and B). In mice, minocycline (30 mg/kg; i.p.) or pentoxifylline (20 mg/kg; i.p.); administered according to the same schedule as in rats improved the response to morphine (10 mg/kg; i.p.) as demonstrated by the von Frey and cold plate tests (Fig. 3C; E and D; F). 3.2.2. The influence of glial inhibitors on morphine intrathecal administration in rats and mice Minocycline (30 mg/kg; i.p.) administered preemptively (16 h and 1 h before CCI) and then twice daily for 7 days in rats with implanted catheters also attenuated the development of allodynia and hyperalgesia (Fig. 4A and B), but did not enhance significantly the response to morphine (30 μg; i.t.) as measured with von Frey test and cold plate test. The effect of morphine (30 μg; i.t.) in minocycline-treated CCI mice was significantly increased as measured by the von Frey test and cold plate test in comparison with vehicle-treated CCI mice injected with morphine (Fig. 4C and D). 4. Discussion Fig. 4. The influence of preemptive and repeated administration of minocycline or pentoxifylline on the effect of a single intrathecal dose of morphine (30 μg; i.t.) on day 7 after CCI in rats and mice. In rats, von Frey (A) and cold plate (B) tests were conducted 60 min after the last dose of minocycline (MC; 30 mg/kg; i.p.; 16 h and 1 h before CCI and then for 7 days twice daily). Morphine (30 μg; i.t.) was injected following the tests and the same tests were repeated 30 min thereafter. In mice, von Frey (C) and cold plate (D) tests were conducted 30 min after the last dose of minocycline (MC; 30 mg/kg; i.p.; 16 h and 1 h before CCI and then 7 days twice daily). Following tests morphine (30 μg; i.t.) was injected and the same tests were repeated 30 min thereafter. The data are presented as the mean ± S.E.M. (8–16 animals per group). Inter-group differences were analyzed by ANOVA Bonferroni's Multiple Comparison Test; ⁎ P b 0.05; ⁎⁎ P b 0.01; ⁎⁎⁎ P b 0.001 indicates a significant difference compared with control (vehicletreated CCI animals or vehicle-treated CCI animals which received a single dose of morphine). In our experiments, we observed the influence of preemptive and repeated systemic administration of minocycline on the development of allodynia and hyperalgesia in rats and mice after CCI. It has been reported that CCI model mimics some characteristic features of neuropathic pain in humans (Bennett and Xie, 1988). Such effect has been already described after propentofylline (a glial inhibitor belonging to xanthine derivatives) treatment in rat model of neuropathic pain (Sweitzer et al., 2001; Raghavendra et al., 2003, 2004); and in the present paper we demonstrate such effects after pentoxifylline (a non-specific cytokine inhibitor) administration in mice after CCI. The study of Raghavendra et al. (2003) showed that intraperitoneal administration of minocycline attenuated the development of behavioral hypersensitivity after L5 nerve transection in rats but had no effect on the treatment of existing mechanical allodynia and hyperalgesia, in spite of significant inhibition of microglial activation in this model (Raghavendra et al., 2003). That was the reason why in J. Mika et al. / European Journal of Pharmacology 560 (2007) 142–149 our study we used preemptive and repeated treatment with minocycline and pentoxifylline to examine their influence on development of neuropathic pain. We observed that such scheme of administration was effective in preventing development of neuropathic pain in CCI rat and mice models. Many papers evidenced by biochemical methods that the doses of minocycline which we used in our study induced glial inhibition (Raghavendra et al., 2003; Li et al., 2005; Ledeboer et al., 2005). It seems that the activated glial cells in the spinal cord release proinflammatory cytokines and other substances thought to facilitate pain transmission (Eriksson et al., 1993; Wagner et al., 1995; Liu et al., 1995; Colburn et al., 1997, 1999; Coyle, 1998; Watkins et al., 2001; Watkins and Maier, 2003). Therefore, microglia might be responsible for the initiation of neuropathic pain states (Eriksson et al., 1993; Kreutzberg, 1996; Marchand et al., 2005). For that reason, preemptive treatment with glial inhibitors seems to be more effective than their administration only after glial cells had already been activated (Raghavendra et al., 2003; Dorazil-Dudzik et al., 2004). Recently, Piao et al. (2006) described the molecular mechanisms underlying the action of minocycline. It seems that minocycline reduced microglial activation by inhibiting p38 mitogen-activated protein kinase (MAPK) in microglia. It has been suggested that the analgesic effects of minocycline in a rat model of neuropathic pain result from: i) an attenuated expression of mRNA for interleukin (IL)-1beta, TNF-alpha, IL-1betaconverting enzyme, TNF-alpha-converting enzyme, IL-1 receptor antagonist and IL-10 in lumbar dorsal spinal cord, ii) reduction of IL-1beta and TNF-alpha protein level in the cerebrospinal fluid, iii) reduction of IL-6 protein level in serum (Ledeboer et al., 2005; Zanjani et al., 2006). These studies prove the hypothesis that antihyperalgesic and antiallodynic effects of minocycline are mediated by microglial cells and are distinct from antimicrobial action of this drug (He et al., 2001; Kloppenburg et al., 1995). Moreover, there are some studies showing that minocycline treatment is neuroprotective in animal models of spinal cord injury (Stirling et al., 2004) and ischemia (Yrjanheikki et al., 1998, 1999). Therefore significant facilitation of motor function recovery, observed in our study, may, at least partly, result from neuroprotective action of minocycline, besides its antiallodynic and antyhyperalgesic action. Our observations are in agreement with other studies showing that minocycline reduces the neurodegeneration observed in brain ischemia, and Huntington's and Parkinson's diseases (Tikka and Koistinaho, 2001; Wu et al., 2002; Yrjanheikki et al., 1998, 1999). Resistance to morphine is characteristic of neuropathic pain (McQuay, 2002; Porreca et al., 1998). The mechanisms of a decreased analgesic potency of morphine in neuropathic pain are not fully understood (Przewlocki and Przewlocka, 2001, 2005). It was suggested that lower effectiveness of morphine in neuropathic pain might be due to the reduced number of presynaptic opioid receptors following degeneration of primary afferent neurons caused by nerve damage (Porreca et al., 1998). Although the role of glia and cytokines mainly in the initiation and maintenance of pain states has been reported (Eriksson et al., 1993; Kreutzberg, 1996; Coyle, 1998; DeLeo and Yezierski, 2001; Watkins et al., 2001, 2005; Watkins and Maier, 2003; Marchand et al., 2005; 147 Ledeboer et al., 2005), much less is known about an influence of modulation of glial activity on analgesic effects of morphine in neuropathic pain. Raghavendra et al. (2002, 2003, 2004) have suggested that the activation of microglial cells and enhanced proinflammatory cytokine expression in the spinal cord have been implicated in the development of morphine tolerance. It is already known that microglia release proinflammatory cytokines (IL-1, IL-6 and TNFalpha) in response to morphine, thereby opposing its effects (Raghavendra et al., 2002, 2003, 2004; Johnston and Westbrook, 2005). In our study, the significant increase in morphine (i.p.) effects was observed on day 7 after preemptive and repeated administration of minocycline (i.p.) following CCI in rats and mice. The same significant potentiation was observed after morphine administration by lumbar puncture in minocyclinetreated mice, but only a tendency was observed in rats. The reason is a different procedure of i.t. administration in rats and mice. The rats were chronically implanted with intrathecal catheters; and morphine was administered i.t. on 12th day after catheter implantation, which itself induces glial activation (DeLeo et al., 1997). We started to administer minocycline 5 days after i.t. catheter implantation before sciatic nerve ligation. However, it is already known that even slight activation of spinal glial cells after catheter implantation in the spinal cord region where morphine is administered can be the reason of the partial loss of morphine effectiveness, which will be further attenuated by the spinal glial activation after CCI (Colburn et al., 1997; Raghavendra et al., 2002; Johnston and Westbrook, 2005). In fact, the allodynia and hyperalgesia are more pronounced in CCI rats with implanted catheters than in CCI rats without catheter implantation (11.5 g ± 0.6 versus 13.5 g ± 0.9 and 4.6 g ± 0.4 versus 6.7 g ± 0.4; respectively). This suggests that even a slight activation of spinal glial cells before CCI and minocycline treatment influences the CCI-induced effect. That may be the reason why we observed an improvement of analgesic effect of intrathecal morphine administration in mice when morphine was injected directly to the spinal cord by lumbar puncture (Hylden and Wilcox, 1980), but only a non-significant tendency was seen in catheter-implanted rats. The results of our experiments support the concept that modulation of glial and neuroimmune activation may be a potential therapeutic target in prevention or treatment of neuropathic pain. Further, restoration of the analgesic activity of morphine by glial inhibitors (minocycline and pentoxifylline) suggests that the increased glial activity and proinflammatory cytokine responses may account for the decreased analgesic efficacy of morphine observed in the treatment of neuropathic pain. 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