The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients

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THE EFFECT OF PENTOXIFILINE ON POST-INJURY HYPEHALGESIA IN RATS AND POSTOPEHATTVE PAIN IN PATIENTS Jerzy Wordliczek’, Antoni M. Szczepanik*, Marcin Banach’, Jadwiga Turchan4, Marek Zembala3, Maciej Siedlar3, Ryszard Przewlocki4, Wojciech Serednicki’, Barbara Przewlocka4 ‘Department of Anaesthesiology and Intensive Care, *I Department of General and GI Surgery, 3Department of Clinical Immunology and Microbiology Collegium Medicum Jagiellonian University, Krakow, 4Department of Molecular Neuropharmacology Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland. (Received in final form October 22, 1999)

Summary

Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre-injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFol level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFo and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor. Key Words:

preemptive analgesia, nociception, cytokines, pentoxifiline, animal study, clinical study

Tissue injury is usually followed by alterations in thermal and mechanical sensibility of the affected area of the body. It has been proposed that surgical injury may lead to comparable alterations in sensory processing, resulting in the amplification and prolongation of postoperative pain. It was suggested (I), that it may be preferable to prevent postoperative pain as well as its neurophysiological and biochemical consequences, rather than to treat pain when these consequences have already been established. In experimental or clinical studies different substances like opioids, local anesthetics and/or NSAID’s have been administered before the experimental injury or surgery in order to prevent or reduce the development of hyperexcitability of the nervous system in the postoperative period. Hence the term - preemptive analgesia. Corresponding author: Barbara Przewlocka Ph.D., Institute of Pharmacology PAN, 12 Smetna str.3 l-343 Krakow, POLAND, e-mail: [email protected], Fax: +48 12 6374500

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Tissue injury induces a cascade of cellular reactions at the lesion area, with an accompanying release, among others, of prointlrunmatory cytokines, such as TNFo, Il-lp, Il-6 and 11-S(2,3,4, 5, 6, 7, 8) which is followed by subsequent inflammatory reactions. Further, cytokines induce the release of pain mediators, such as bradykinin (9) and CGRP (10) and activate vagal afferents (11, 12). In consequence, injury, by the release of cytokines can produce a long-lasting hyperalgesia. In the formalin test, an experimental model of hyperalgesia, two sensitization mechanisms are present: in phase I sensitization occurs peripherally, while in phase II it occurs centrally. It is thought that cytokines are important mediators in both phases. Consequently, it may be proposed that the systemic administration of cytokine inhibitors may hinder both sensitization phases, while central administration may impede the process of central sensitization. Thus, the purpose of our study was to examine whether inhibition of cytokine synthesis beginning before injury could inhibit the development of hyperalgesia. Pentoxifiline (PTFL), a drug that can hinder the production of cytokines, was chosen for our study. The choice of PTFL as a nonselective cytokine synthesis inhibitor was dictated by the possibility of using PTFL both in animal and clinical studies. The drug inhibits the production of TNP(r by blocking the transcription of the gene responsible for TNTa synthesis. As a non-specific inactivator of phosphodiesterase, it increases the CAMP level in cells, inhibiting not only the synthesis of TNFa, but also ofIL-ll3, IL-6, and IL-8 (13, 14, 15, 16). In an experimental model, we investigated the influence of pre-injury PTFL administration on pain thresholds and pain behaviour. The TNFa level in serum was also measured. Our clinical investigation focused on the influence of preemptive PTFL administration on visual analoque scales of pain intensity (VAS), opioid requirement, and serum cytokine levels in the early perioperative period. Material and methods Animal study

Male Wistar rats, weighing 250-320 g each, were housed in groups of eight to a cage on a constant light-dark cycle (the light on between 08.00 and 20.00 h), with unrestricted access to food and water. In order to mimic the nociceptive stimulation that takes place during surgical procedures, the formalin was employed. 15 minutes after baseline measurement of nociceptive threshold and preformalin intraperitoneal (i.p.) or intrathecal (it.) administration of PTFL or saline, rats were lightly anesthesized with halothane (l-2 ~01%) and 100 ~1 of 10% formalin solution was injected subcutaneously fi. c.J into the dorsal surface of the hind left paw (17, 18). Halothane was used for ethical reason to eliminate the painfbl effect of formalin injection and to parallel a clinical surgery procedure. Other studies showed the lack of the antinociceptive effect of halothane on the formalin-induced facilitation (18, 19). The influence of pre-injury i.p. PTFL (0.1, 1.O, or 10 mg/kg, n=30; 10 rats for each dose) or i.t. PTFL (150 l.rg, n=lO) administration on pain-related behaviour, was quantified by counting spontaneous flinches, shakes and jerks of the formaline-injected paw. All such behaviours are referred to as jerks in the text. Paw jerks were continuously counted for each individual animal for 55 min and then scored for four characteristic time points: O-5 min (first phase of inflammatory response induced by formalin) and 40-45, 45-50, 50-55 min (second phase of inflammatory response) after formalin administration. The nociceptive threshold for mechanical stimuli was evaluated using the paw pressure test (PP test). The animal was gently restrained and an incremental pressure was applied via a piston onto the dorsal surface of the hind paw. The cut-off pressure was 480 g. The measurements were taken 3 times at 15- or lo-second intervals, and their mean was used for calculations.

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The effect of PTFL given alone, on the nociceptive threshold for mechanical stimuli was measured in rats 30 and 55 min abler the i.p. administration of PTFL (0.1, 1.O, or 10 mg/kg, n=30; IO rats for each dose ) or i. t. PTFL (150 pg, n=lO) injection. Experiments with i.t. administration of PTFL or saline were conducted on rats with a chronically implanted catheter (PE 10, Clay Adams) in the subarachnoid space at the rostra1 level of the lumbar enlargement according to Yaksh and Rudy (20). In subsequent experiments rats received pre-formalin injection of PTFL i.p. (0.1, 1,O, or 10 mg/kg, n=30; 10 rats for each dose). The control group (n=lO) was administered i.p with saline. Rats with chronically implanted i.t. catheters were injected prior to formalin administration with i.t. PTFL (150 I.rg in a volume of Spl, n=lO) and control (n=lO) rats received i.t. saline. 55 minutes after formalin injection, the nociceptive threshold for mechanical stimuli was measured by the PP test. The effect of i.p. administration of PTFL (lOmg/kg, n=lO) before formalin on antinociceptive effects of opioids in rats was determined using morphine (1 mg/kg) administered s. c. 55 min after the formalin injection. Controls (n=lO) received saline instead of PTFL. The nociceptive threshold for mechanical stimuli (PP test) was measured 15 and 30 min after morphine administration. Two groups of rats were also employed for biochemical studies: the PTFL group (n= 40, 10 rats for each measurement), which received i-p. PTFL (10 mg/kg) 15 min before formalin injection and the control group (n= 40, 10 rats for each measurement), which received i.p. saline and 15 min later formalin. At 10, 30, 60 or 90 min after the formahn injection the rats were decapitated and trunk blood was collected. The BioSource International CytoscreenTM Rat Tumor Necrosis Factor-Alpha (raTNF-ol) ELISA kit was used for the TNF-a serum level determination. The experiments were carried out according to the protocol approved by the Ethical Commission of the Institute of Pharmacology. Clinical study. 34 patients scheduled for elective open cholecystectomy were studied. This operation was chosen

on account of its uniform duration, course of surgery and magnitude of surgical trauma, which allows for repeatable data to be collected. Moreover, cytokine profile in serum in the postoperative period following open cholecystectomy has already been described, which facilitated planning the timing of blood sampling (21). Preoperatively none of the patients had acute and/or chronic intlammatory processes. Patients were randomly allocated into the PTFL (n=19) and control (n=15) groups. In PTFL group the average age was 52.8 years (spread 36-59) while in control group the average age was 5 1.3 years (spread 39-57). The average duration of procedure was 83 minutes (65-95) in the former and 87 min (50-105) in the latter. Operations were performed under general anesthesia. Patients were administered PTFL (10 m&g) in IO0 ml 0.9% NaCl or an equal volume of placebo 30 minutes before the operation. Following the operation, patients were transferred to the recovery room and postoperative analgesia was administered via a Patient-Controlled Analgesia (PCA) system, to enables patients to self-administer doses of analgetics. After the activation of PCA patients were given boluses of 20 mg of pethidme with a lockout time of 5 min. The total pethidine requirement was determined in the early postoperative period (24 hours), for all patients. The level of intensity of postoperative pain was also measured using the Visual Analoque Scale (VAS) with measurements at three times: VA& - pain intensity which made the patient activate the analgesic system, VA& - after 6 hours following the operation and VA& after 24 hours following the operation. Blood samples were also collected 1 h before the operation and 2, 4, 8, 12, and 24 hours after skin incision. Serum levels of TNF-a, ILlJ3 and IL6 were determined using ELISA kits (The BioSource International Cytoscreen TM ). The clinical investigation was carried out according to

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the protocol University.

approved

by the Ethical

Commission

of the Collegium

Medicum

Jagiellonian

Data analysis The results were assessed by an analysis of variance (ANOVA). Inter-group differences were analysed by Duncan’s multiple-range test. Biochemical and animal data are presented as means f SEM, clinical data are presented as means f SD. Results

Animal stu@ PTFL injected alone i.p, or i.t., has no significant effect on pain threshold for mechanical stimuli at the times studied, neither as compared with the initial measurement nor with the control group (data not shown). After injection of formalin hyperalgesia was observed up to 60 minutes, however sign&ant decrease in nociceptive threshold was observed 55 minutes after formalin only in group of rats with intrathecal catheters (Fig. IA, B control rats). PTFL administered i.p. in doses of 1.0 or 10 mgkg, 15 minutes before the injection of formalin significantly increased (p < 0.05) the pain threshold for mechanical stimuli measured 55 minutes after the injection of formalin, compared to the control group (Fig. IA.). The statistically significant increase (p < 0.05) in the pain threshold was also observed with i.t. injection of PTFL 15 minutes before the injection of formalin (Fig. IB). A.

B. *

250

200 150

150 F!

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100

50 --

50

0

a1-r baseline

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0.1 mgRg

t mgncg

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Fig. 1 The effect of PTFL: A.- (0.1, 1.0, or 10 mg/kg, i.p.), B.- (150 M, i.f.) administered 15 minutes before the xc. injection of 10% formalin solution, on the nociceptive threshold investigated in rats in the paw pressure test 55 minutes following the formalin injection (mean f S.E.M.), * indicates statistical significance @x0.05) related to the control group, # indicates statistical significance (lxO.05) related to the baseline measurement. PTFL administered i.p. in doses of 0.1 and 1.O mgkg, 15 minutes before the injection of formalin had no influence on the pain behaviour induced by formalin at any time intervals. However, a higher dose of PTFL (10 mgkg) decreased the number of formalm-induced symptoms at all time

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intervals (p < 0.05) (Fig. 2A). A similar effect was observed with i.t. injection formalm but only in the second phase (40-55 mm) (Fig. 2B).

2

T

= 50 ;

40

$

30

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50--

E 2 20 Y 10 0 510.

4045

45-50

50-55

5-10.

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40-45

45-50

50-55

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Fig. 2 TheeffectofPTFL: A.-(0.1, 1.0, or lOmg/kg, i.p.), B.-(150 pg, Lt.) onthedevelopment of pain behaviour induced by 10% formalin solution adminktered s. c. 15 minutes after PTFL injection (mean f S.E.M.), * indicates statistical significance 0.05) related to the control group at the relevant point of times Morphine (1 mg/kg) administered xc. 55 minutes after the injection of formalin, causes a significant increase in the pain threshold for mechanical stimuli at both time intervals as compared with the baseline level. Administration of PTFL (10 mg/kg) i.p. enhanced the antinociceptive effect of morphine in the formalin model. Nociceptive thresholds were significantly higher (p < 0.05) in the PTFL group in comparison with the control group at both time intervals following morphine administration (Fig. 3). n control (saline) i.p. + MOR

*

a PTFL lOma/ka i.D. + MOR

baseline

15

time [min]

Fig. 3 The effect of PTFL (10 mgkg) administered i.p. 15 minutes before the injection of 10% formalin solution, on the nociceptive threshold investigated in rats in the paw pressure test 15 and 30 minutes following morphine (MOR) administration. Morphine (1 mg/kg) was injected S.C.55 minutes following the formalin administration (mean f S.E.M.), * indicates statistical significance (pCO.05) related to the control group, # indicates statistical significance (p~0.05) related to the baseline measurement.

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Administration of formalin into the rat hind paw evokes a significanst increase in the TNFa level, that peaks 60 minutes later. Administration of PTFL before formalin injection (PTFL group) significantly (p < 0.05) inhibits the increase in the level of TNFJa, evident 60 minutes after formahn injection (Fig. 4)

W control (saline) i.p.

‘iI 2

80 60 40 20 0 baseline

10

30

60

90

time [ min ]

Fig. 4 The effect of PTFL (10 mgikg) administeredi.p. on the increase in the level of TNFa induced by

the admiuistration

of 10% foxmalin solution 15 min after PTFL injection

(mean f S.E.M.), * indicates statistical significance (pCO.05)related to the control group at the relevant point of time. Clinical study

Pethidine requirements in the early postoperative period were significantly lower @ < 0.05) in patients who were administered of PTFL i.v. (PTFL group) and was 213.7 f 32.7 mg (160 mg 280 mg) as compared with 299.1 f 44.3 mg (200 mg - 380 mg) in the control group. However, the postoperative pain intensity did not differ between PTFL and control groups (Fig. 5). These data indicate that patients self administered a smaller amount of pethidine to achieve a similar level of analgesia.

w control (saline) i.v.

VAS 3

PTFL 10 mglkg

-I 0

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Fig. 5 The effect of preemptivei.v. administrationof PTFL (10 mgikg)on the postoperative pain level in the early postoperativeperiod (meanf SD.).

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For a majority of patients, the preoperative level of TNFa was undetectable. The increase in serum TNFa was observed at 8, 12 and 24 h after the surgery, but at 8 h and 12 h TNFa levels were significantly (p < 0.05) lower in the PTFL group (Fig. 6).

0

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time [ hours ]

Fig. 6 The effect of the preoperative i.v. administration of PTFL (10 mgkg) on the level of TNFa in the early postoperative period (mean f S.E.M.), * indicates statistical significance@<0.05)related to the control group at the relevantpoint of time. In most patients of both groups, in the scheduled time points IL-lp level was undetectable, and no difference between groups was observed. IL-6 levels in both groups at the start point were similar. However, at 8 and 12 h after surgery, IL-6 serum levels in the control group were significantly higher (p < 0.05) than in the PTFL group. At 24 hours after surgery IL-6 levels were as presented below (Fig. 7). There were no adverse events related to the administration of PTFL.

0

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time [ hours ]

Fig. 7 The effect of preemptive i. v. admindation of PTFL (10 mgkg) on the level of IL-6 in the early postoperative period (mean f S.E.M.), * indicates statistical significance @<0.05) related to the control group at the relevant point of time. Discussion

The present study demonstrates that i.p. or it. PTFL injected alone in rats does not influence the nociceptive threshold for mechanical stimuli. Thus PTFL has no specific direct antinociceptive action in animals, consistent with results obtained in patients complaining of vascular diseases

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who were regularly treated with PTFL, and in whom no direct analgesic effect of PTFL was observed (22). However, injection of PTFL, i.p. or i.t. before formalin administration, increased the nociceptive threshold for mechanical stimuli in rats. This effect of PTFL may be related to a reduced inflammatory response, which was observed after preemptive PTFL administration in the carrageenan inflammation model (23). The increased pain thresholds observed in the present study tier pre-injury PTFL also correspond with the finding that the development of hyperalgesia after formalin administration is inhibited in animals given i.p. IL- 1 receptor antagonists (IL- 1 ra) ( 12). The present result suggest that prior injection of PTFL 10 mg/kg i.p. reduces pain-related behaviour in phase I of the formalin-induced pain model, by inhibiting cytokine activation reducing the in&unmatory response and the process of peripheral sensitization. In phase II, preinjury PTFL administered i.p. or i.t., by inhibiting cytokine activation in the central nervous system, presumably reduces the development of central sensitization. The absence of inhibitory action of pre-injury PTFL administered i.t. on the occurrence of pain-related behaviour in phase I (peripheral sensitization) may be related to the action of i.t. PTFL being limited to central nervous system structures. The enhancement of the antinociceptive effects of morphine observed in our study after the pre-injury i.p. PTFL administration may result from both the inhibition of peripheral and central sensitization as well as the modulatory effects of PTFL on the expression and the release ofnociceptive mediators. It is known that glutamate, aspartate, and substance P released in the spinal cord in response to formalin are critical in producing hyperalgesia as measured either by the nociceptive threshold test or by symptoms of pain-related behaviour (24, 25, 26). However, although the release of substance P and excitatory amino acids within spinal cord is critical for formalin hyperalgesia to occur, their release does not directly produce it. These mediators initiate cascade of events, culminating in the release of substances more directly causal to hyperalgesia, such as cytokines, NO, NMDA and cyclooxygenase products (27). Consequently, the antinociceptive effect of preinjury PTFL on formalin-induced pain, may be explained in terms of the inhibition of post-injury cytokine cascade, since the key role of cytokines in the development of hyperalgesia has already been established (4, 5, 6, 7, 8, 28, 29, 30, 3 1). Intraventricular or i.t. administration of cytokines is already known to cause hyperalgesia (12). Likewise, peripheral bacterial lipopolysaccharide (LPS) administration or injury cause the activation of cytokines not only in peripheral tissues, but also in the cerebrospinal fluid (32). This confirms the potential inhibitory effect of PTFL that hampers cytokine activation in the central nervous system, especially following i.t. administration. It is also established that during the inflammatory response in the peripheral nervous system the gene responsible for coding cytokine IL-lp (a strong iNOS modulator) switches on, causing the NO pronociceptive neurotransmitter level to increase in brain tissue and the cerebrospinal fluid, thereby leading to the development of hyperalgesia (28, 29, 30, 31). Moreover, it was established that PTFL inhibits the production of other proinflammatory cytokines, such as TNFu and IL-6, which are also activated by an injury or a surgery (13). Some researchers report that PTFL can also reduce the production of IL-l (13), which may serve to explain our present observation of the inhibitory effect of pre-injury PTFL administered i.t., on hyperalgesia, which was observed in the present study, especially in view of the fact that there may also exist other sources of cytokine synthesis in the central nervous system, i.e. microglia or astrocytes (27). The results of experimental investigations discussed above tilly justify clinical trials. For ethical reasons, the development of nociception in the postoperative period was determined through opioid administration by PCA. Additionally, postoperative pain intensity was measured in order to find possible differences between groups in terms of pain sensitivity as well as pain intensity 6 and 24 hours following the operation. The similarity of VAS results in both groups testifies to a comparable quality of pain self-management in those patients. The same pain level scored by VAS

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after lower opioid dose used by patients treated preemptively with PTFL, imply that this drug, although has no specific analgesic properties, modulates nociception in early postoperative period. It confirms that reduced release of the inflammatory transmitters may lower the use of analgetics after surgery. Likewise, the results of our recent investigations have confirmed a beneficial postoperative effect of PTFL administered before and after the major oncological surgery (33). Our experimental study also demonstrates that pre-injury PTFL, probably via the inhibition of TNFa release, reduces pain-related behaviour induced by experimental infhunmation, accompanied by a decrease of TNFa serum level, which reinforces the hypothesis of an inhibitory effect of PTFL on nociception. In our clinical study, along with the reduced opioid requirement, significantly lower serum levels of TMa and IL-6 were also observed. The time dependence of the effects in animals and humans differed for several reasons. First, a considerably shorter-lasting anesthesia in animals (2-3 min) was applied in comparison with human surgery (83-87 min), which may have influenced the clearence of the drug. Additionally, the half-life of different drugs in the rat is a few times shorter than in humans, which reflects a faster metabolic rate in the rat. Our results support the hypothesis of an indirect antinociceptive effect of PTFL via the inhibition of pronociceptive cytokine synthesis. The modulation of nociception by an inhibitor of cytokine synthesis may have widespread clinical implications. It increases the options for effective postoperative pain treatments that may reduce opioid requirement and decrease the risk of opioid side effects. Preemptive analgesia constitutes a rational mode of PTFL use as a cytokine inhibitor, since it prevents their synthesis even before their cascade has been lily activated. PTFL used in this way may influence not only nociception itself, but also reduce the general systemic response to an injury, which may result in shorter postoperative recovery, earlier discharge and fewer complications. Consequently, the above data justify the continuation of broadly conceived research into the role of cytokine response modulation in the perioperative period. Acknowledgements

The study was supported by a grant for The State Committee for Scientific Research, KBNWarszawa No. 4 POSC 006 16. The authors would like to thank Professor T. Popiela, Head of 1-st Department of General and GI Surgery Collegium Medicum Jagiellonian University for helpful advice and encouragement during the realisation of this study. References

1. P.D. WALL, Pain, 33 289-290, (1988). 2. J. LUBER-NAROD, R. KAGE, S.E. LEEMAN, J. Immunol., 152 8 19-824, (1994). 3. J.R. GORDON, S.J. GALLI, Nature, 346 274-276, (1990). 4. F.Q. CUNHA, POOLE S., B.B. LORENZETTI, S.H. FERREIRA, Br. J. Pharmacol., 107 660-664, (1992). 5. C.J. WOOLF, A. ALLCHORNE, B. SAFIEH-GARABEDIAN, S. POOLE, Br. J. Pharmacol. 121417-424, (1997). 6. T. OKA, K. OKA, M. HOSOI, T. HORI, BrainRes., 692 123-128, (1995). 7. L.R. WATKINS, L.E. GOEHLER, J. RELTON, M.T. BREWER SF. MAIER, Brain Res. 692 244-250, (1995). 8. L.S. SORKIN, W-H. XIAO, R. WAGNER R.R. MYERS, Neuroscience, 81 255-262, (1997). 9. M.N. PERKINS, D. KELLY, A.J. DAVIS, Canad. J. Physiol. Pharmacol., 73 832-836, (1995).

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10. X-Y. HUA, P. CHEN, A. FOX, R.R. MYERS, Journal of Neuroscience, 16 4742-4748, (1996). 11. M. LESHNER, L.E. GOEHLER, J. HERMANN, J.K. RELTON, S.F. MAIER L.R. WATKINS, Brain Res. Bulletin, 37 605-610, (1995). 12. L.R. WATKINS, E.P. WIERTLAK, L.E. GOEHLER, K.P. SMITH, D. MARTIN, MAIER S.F., Brain Res., 654 15-26, (1994). 13. P. NEUNER, G. KLOSNER, E. SCHAUER M. POURMOJIB, W. MACHEINER, C. GRUNWALD, R. KNOBLER, A. SCHWARZ, T.A. LUGER, T. SCHWARZ, Immunology, 83 262-267, (1994). 14. J. SEMMLER, U. GEBERT, T. EISENHUT, J. MOELLER, M.M. SCHONHARTING, A. ALLERA, S. ENDRES, Immunology, 78 520-525, (1993). 15. P. ZABEL, F .U. SCHADE, M. SCHLAAK, S. MURPHY, Immunobiology, 187 447 463, (1993). 16. J. HAN, P. THOMPSON, B. BEUTLER, J. Exp. Med., 172 391-394, (1990). 17. T.J. CODERRE, M.E. FUNDYTUS, J.E. McKENNA, S. DALAL, R. MELZACK, Pain, 54 54-60, (1993). 18. E.S. FU, J.E. SCHARF,Reg. Anesthesia, 20 217-221, (1995). 19. T. GOTO, J.J. MAROTA, G. CROSBY, Anesthesiology, 80 409-416, (1994). 20. T.L. YAKSH, T.A. RUDY,Physiol. Behav., 17 1031-1036, (1976). 21, J.M. BELLON, L. MANZANO, L. BERNARDOS, N.G. HONDUVILLA, A. LARRAD, J. BUJAN, M. ALVAREZ-MON, Eur. Surg. Res., 29 27-34, (1997). 22. C.P. SAMLASKA, E.A. WINFIELD, J. Am. Acad. Dermatol., 30 603-621, (1994). 23. Y.L. CHEN, V. LE VRAUX, J.P. GIROUD, L. CHAUVELOT-MOACHON, Eur. J. Pharmacol., 271 319-327, (1994). 24. D.H. SMULLIN, S.R. SKILLING, A.A. LARSON, Pain, 42 93-101, (1990). 25. E.P. WIERTELAK, L.E. FURNESS, R. HORAN, J. MARTINEZ, S.F. MAIER, L.R. WATKINS, Brain Res., 649 19-26, (1994). 26. C.W. MURRAY, A. COWAN, A.A. LARSON, Pain, 44 179-185, (1991). 27. L.R. WATKINS, D. MARTIN, P. ULRICH, K.J. TRACEY, S.F. MAIER, Pain, 71 225-325, (1997). 28. M.-L WONG, P. BONGIORNO, V. RETTORI, S. MCCANN, J. LICINIO, Proc. Natl. Acad. Sci. USA, 94 227-232, (1997). 29. S. LIU, L.M. ADCOCK, R.W. OLD, P.I. BARNES, T.W. EVANS, Biochem. Biophys. Res. Commun., 196 1208-1213, (1993). 30. R.A. ROBBINS, D.R. SPRINGAL, J.B. WARREN, O.J. KWON, L.D.K. BUTTERY, A.J. WILSON, L.M. ADCOCK, V. RIVEROS-MORENO, S. MONCADA, J. POLAK, P.J. BARNES, Biochem. Biophys. Res. Commun., 198 835-843, (1994). 31. D. GRZYBICKI, G.F. GEBHART, S. MURPHY, Journal of Chemical Neuroanatomy, 10 221-229, (1996). 32. F. COCEANI, J. LEES, C.A. DINARELLO, Brain Res., 446 245-251, (1988). 33. J. WORDLICZEK, A.M. SZCZEPANIK, H. WIATRAK, J. BUCKI, T. POPIELA, Shock, 7 (suppl.) 95, (1997).