Endotoxin-induced local inflammation and hyperalgesia in rats and mice: a new model for inflammatory pain.

Lipopolysaccharide, also known as endotoxin (ET), is a major constituent of the outer membrane of the cell wall of most gram negative bacteria. ET is known to cause a number of pathophysiological changes associated with illness including inflammatory pain. The aim of this study is to characterize the peripheral hyperalgesia induced by ET in rats and mice. Different groups of rats and mice received different doses of ET ranging from 0.6 microgram to 40 micrograms dissolved in 50 microliters saline and injected in the plantar area of the left hind legs. All animals were subjected to tail immersion (TF), hot plate (HP) and paw pressure (PP) tests, 2-3 days prior to ET injection and during the following 1-2 days. ET injections produced a dose-dependent decrease in the latencies of the HP and PP tests of the injected leg reaching a maximum decrease of 50-60% of the control with 20-40 micrograms ET at 9 h (rats) and 24 h (mice) after the injection. Almost complete recovery was observed after 24 h in rats and 48 h in mice. TF latencies showed a less but a significant decrease while PP of the opposite leg and all tests in saline-injected animals did not elicit significant variations and served as additional controls. Our results indicate that the use of ET-produced hyperalgesia is a valid model for local and reversible inflammatory pain, with minimal distress to the animal. This model can also be used to study the efficacy of various anti-inflammatory and analgesic drugs and the molecular mechanisms of inflammation induced by bacterial invasion.

Functional interplay between gelatinases and hyperalgesia in endotoxin-induced localized inflammatory pain.

The role of ECM-degrading proteinases in normal developmental processes and in pathological conditions is extensively studied. However, few reports describe the role ECM-degrading proteinases play in modulating hyperalgesia. The goal of this study is to describe the regulation of gelatinases during endotoxin mediated local inflammation, induced by intra plantar endotoxin (ET; 1.25 microg/50 microl) injection in Balb/c mice, and to correlate that with hyperalgesia. ET injections induced hyperalgesia, as determined by hot plate and paw pressure tests, which peaked by 24 h and recovered by 48 h post-injection. Contralateral paw of ET injected mice and saline injected paws in control mice elicited no hyperalgesia. Zymography showed that ET and saline injected paws elicited increased gelatinase activity by 9 h after injection. However, only the former maintained high levels of expression of a 90 kD gelatinase up to at least 96 h post ET injection, while in the latter gelatinase expression was down regulated by 24 h. Interestingly, the 90-kD gelatinase was upregulated in the contralateral paw of the ET-injected mice beyond 48 h post injection. Saline injection in that paw, during a time when gelatinases are upregulated, induced hyperalgesia. Intraperitoneal injection of either ZnCl(2) (100 microM), thymulin (5 microg/100 microl), or morphine (2 mg/kg/100 microl) reversed the ET-induced hyperalgesia and suppressed gelatinase activity. Furthermore, intraperitoneal injection of MPI, an ECM-degrading proteinase inhibitor, reversed ET induced hyperalgesia. Taken together, the above suggests that a functional interplay exists between gelatinase upregulation triggered by ET injections and hyperalgesia. The exact mechanism underlying such correlation remains to be determined.

Zinc reduces the hyperalgesia and upregulation of NGF and IL-1 beta produced by peripheral inflammation in the rat.

The effect of systemic zinc administration on the inflammatory hyperalgesia induced by intraplantar injections of either complete Freund's adjuvant (CFA) or bacterial endotoxin/lipopolysaccharide (LPS) in a hindpaw of adult rats was investigated. CFA injection resulted in mechanical and thermal hyperalgesia and an elevation in the levels of interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) in the ipsilateral hindpaw. Zinc treatment (20 nmole) significantly reduced sensitivity in the early phase of the inflammation and diminished the increase in the levels of IL-1 beta and NGF without affecting paw swelling. Intraplantar LPS injection also produced mechanical hyperalgesia and this too was reduced by zinc administration in a dose-dependent fashion (0.1-20 nmoles). Our results indicate that zinc has an analgesic action during early inflammation and that this may be the consequence of reducing levels of the inflammatory cytokine IL-beta and the growth factor NGF.

The role of the sympathetic efferents in endotoxin-induced localized inflammatory hyperalgesia and cytokine upregulation.

The sympathetic system (SNS) is considered to be a major component of the neurogenic contribution to inflammation and hyperalgesia. We have investigated the role of the SNS in the local inflammatory pain induced by intraplantar (i.pl) injections of bacterial endotoxin (ET). Treatment of rats with an alpha-adrenoceptor antagonist (phentolamine, 0.25-1 mg/kg, i.p.), a beta-adrenoceptor antagonist (propranolol, 1-10 mg/kg, p.o.) or a sympathetic neuron-blocking agent (guanethedine, 30 mg/kg, s.c.) resulted in a dose-dependent reduction of the thermal hyperalgesia induced by ET. Mechanical hyperalgesia, however, was less sensitive to inhibition by propranolol and guanethedine but significantly inhibited by phentolamine. ET injection produced significant upregulation of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, and nerve growth factor (NGF). Treatment with any one of the three sympatholytics abolished the upregulation of NGF and IL-6, while phentolamine and guanethedine also reversed the upregulation of TNF-alpha. IL-1 beta was resistant to all of the sympatholytic treatments. We conclude that the SNS can contribute to the local inflammation and hyperalgesia following injection of ET. The resistance to sympatholytics shown by IL-1 beta, known to play a key role in the inflammatory cascade, suggests that ET can initiate inflammation and hyperalgesia independently of peripheral and central sympathetic mechanisms.

The role of cytokines and prostaglandin-E(2) in thymulin induced hyperalgesia.

We have recently reported that intraperitoneal (i.p.) injection of thymulin at low doses (50 ng) resulted in thermal and mechanical hyperalgesia and upregulation of the level of interleukin-1beta in the liver. In this study, we demonstrate that such injections of thymulin result in a significant elevation in the levels of TNF-alpha (P<0.01), NGF (P<0.01) and PGE(2) (P<0.01) in the liver of the treated rats, in addition to the increase in the levels of IL-1beta. Pretreatment with specific antagonists to each of these factors (polyclonal anti-TNF-alpha, anti-NGF antiserum and IL-1 receptor antagonist) did not result in the abolition of the hyperalgesia as assessed by the paw pressure, hot plate, paw immersion and tail flick tests. However, pretreatment with a combination of the above antagonist and antisera almost completely prevented thymulin-induced hyperalgesia. The cyclooxygenase inhibitor, meloxicam, reversed in a dose dependent manner (0.2, 0.4 and 2 mg/kg) thymulin effects as assessed by the different pain tests. It also abolished the thymulin-induced increase in the level of cytokines and NGF in the liver. Our results indicate that PGE(2) could be the key mediator of the hyperalgesic action of thymulin and the observed upregulation of proinflammatory cytokines and NGF.

Interleukin-10 reduces the endotoxin-induced hyperalgesia in mice.

In the endotoxin-induced inflammation, interleukin-10 reduced significantly, and in a dose-dependent manner, the inflammatory pain as assessed by mechanical and thermal tests. The levels of Tumour Necrosis Factor (TNF)alpha and NGF were upregulated at 1.5 h whereas those of IL-1beta at 6 h after ET injection. IL-10 downregulated the levels of TNFalpha (from 4974.75 +/- 875.78 to 1008 +/- 350 pg/hind paw), NGF (from 352.9 +/- 46.7 to 33.9 +/- 2.4 pg/hind paw) and IL-1beta (from 2773.88 +/- 423.96 to 1108 +/- 399.56 pg/hind paw). These data suggest that IL-10 inhibits ET-induced hyperalgesia by downregulation of TNFalpha, IL-1beta and NGF production.

Involvement of capsaicin sensitive primary afferents in thymulin-induced hyperalgesia.

Intraplantar (5 ng) or intraperitoneal (50 ng) injections of thymulin, produced both thermal and mechanical hyperalgesia in rats. In this report, we show that ablation of capsaicin sensitive primary afferents (CSPA) can alter or abolish thymulin-induced hyperalgesia. Different groups of rats were subjected to either treatment with capsaicin or to surgical subdiaphragmatic vagotomy (SDV). Both capsaicin and SDV reduced significantly thymulin-induced hyperalgesia. On the other hand, these treatments elicited differential effects on the modulation by thymulin of the levels of nerve growth factor and interleukin 1beta. We conclude that the hyperalgesic effects of i.p. thymulin are mainly mediated through the CSPA fibers.

Hyperalgesia induced by low doses of thymulin injections: possible involvement of prostaglandin E2.

Thymulin injection into rats (20-150 ng) i.p. caused a significant reduction in both mechanical (paw pressure test) and thermal (hot plate and tail flick tests) nociceptive thresholds. Thymulin injection also doubled IL-1beta level in the liver of these animals. Induced hyperalgesia was reversed completely by alpha-MSH related tripeptide, Lys-D-Pro-Val in low doses, which is known to antagonize IL-1beta and PGE2 induced hyperalgesia, but was only partly reversed by IL-1beta related tripeptide, Lys-D-Pro-Thr at high doses, which is known to antagonize IL-1beta induced hyperalgesia only. We conclude from these results that thymulin causes hyperalgesia and that this effect is at least in part mediated via PGE2 and its effectiveness at low concentration implies a physiological role for this thymic hormone.

Involvement of substance P, CGRP and histamine in the hyperalgesia and cytokine upregulation induced by intraplantar injection of capsaicin in rats.

Intraplantar (i.pl.) injection of small doses of capsaicin has been shown to produce hyperalgesia and upregulation of the levels of proinflammatory cytokines. The present work aimed at investigating the possible mediation of these effects by sensory neuropeptides and mast cells. Various groups of rats received i.pl. injection of capsaicin alone or preceded by the injection of antagonists to substance P (SP), calcitonin gene-related protein (CGRP) and histamine (H1, H2) or the mast cell blocker ketotifen. All pretreatments prevented, in a dose-related manner, the capsaicin-induced hyperalgesia. The SP, H2 antagonists and ketotifen prevented the upregulation of all cytokines and nerve growth factor (NGF) levels, while the CGRP and H1 antagonists showed only attenuation of the NGF level.

Thymulin induces c-fos expression in the spinal cord of rats which is reversed by meloxicam and morphine.

Intraplantar (i.pl.) injections of thymulin have been shown to produce hyperalgesia in rats through a prostaglandin E2-dependent mechanism. This study aimed at investigating if such injections can produce sustained activation of spinal neurons by mapping the fos-like-immunoreactivity (FLI) as a marker for this activation. Our results showed that thymulin produces significant and sustained FLI in neurons located in spinal laminae known to be involved in nociception. Pretreatment with either morphine or meloxicam (a cyclooxygenase inhibitor) revealed differential effects on FLI and the hyperalgesia induced by thymulin. These findings support the hypothesis that thymulin can affect central neurons either directly or through the peripheral nerve terminals.

Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity.

Experimental inflammation produced by an intraplantar injection of complete Freund's adjuvant results in local sensory hypersensitivity and up-regulates the neuropeptides substance P and calcitonin gene related peptide in the primary sensory neurons innervating the inflamed tissue. The inflammation also elevates nerve growth factor levels in the skin. Systemic administration of anti-NGF neutralizing antibodies prevent the behavioral sensitivity, the up-regulation of neuropeptides and the inflammation-induced expression of the immediate early gene c-fos in dorsal horn neurons, without modifying swelling and erythema. Elevation of the neurotrophin NGF in the periphery is a major contributor, therefore, of inflammatory pain.

Thymulin reverses inflammatory hyperalgesia and modulates the increased concentration of proinflammatory cytokines induced by i.c.v. endotoxin injection.

The immunomodulatory thymic hormone thymulin has been shown previously to possess anti-inflammatory actions in the periphery. In this study, we have examined the effect of i.c.v. injections of either endotoxin (ET) or thymulin, in separate groups of conscious rats, on pain-related behavior and cytokine levels in different areas of the brain. Furthermore, we investigated the effect of pretreatment with either i.c.v. or i.p. injections of thymulin on endotoxin-induced hyperalgesia and the effect of pretreatment with i.c.v. thymulin on endotoxin-induced up-regulation of cytokine levels. Our results demonstrate that i.c.v. injection of endotoxin (1 microg in 5 microl saline) resulted in a significant decrease in the nociceptive thresholds as assessed by different pain tests, with peak hyperalgesia at 3 h. However, thymulin at different doses, when injected (i.c.v.), had no significant effect on pain related behavior. Pretreatment (i.c.v.) with thymulin (0.1, 0.5 and 1 microg in 5 microl saline) 20 min before endotoxin (i.c.v.) injection (1 microg in 5 microl saline) reduced, in a dose dependent manner, the endotoxin-induced hyperalgesia and exerted differential effects on the up-regulated levels of cytokines in different areas of the brain. The results provide behavioral and immunochemical characterization of a rat model for intracerebral inflammation and indicates a neuroprotective role for thymulin in the CNS.

Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor alpha.

1. Peripheral inflammation is characterized by heightened pain sensitivity. This hyperalgesia is the consequence of the release of inflammatory mediators, cytokines and growth factors. A key participant is the induction of the neurotrophin nerve growth factor (NGF) by interleukin-1 beta (IL-1 beta). 2. Tumour necrosis factor alpha (TNF alpha) has been shown both to produce hyperalgesia and to upregulate IL-1 beta. We have now examined whether the induction of TNF alpha in inflammatory lesions contributes to inflammatory sensory hypersensitivity by inducing IL-1 beta and NGF. 3. The intraplantar injection of complete Freund's adjuvant (CFA) in adult rats produced a localized inflammation of the hindpaw with a rapid (3 h) reduction in withdrawal time in the hot plate test and in the mechanical threshold for eliciting the flexion withdrawal reflex. 4. The CFA-induced inflammation resulted in significant elevation in the levels of TNF alpha, IL-1 beta and NGF in the inflamed paw. In the case of TNF alpha, an elevation was detected at 3 h, rose substantially at 6 h, peaked at 24 h and remained elevated at 5 days, with similar but smaller changes in the contralateral non-inflamed hindpaw. No increase in serum TNF alpha was detected at 24 h post CFA injection. 5. Intraplantar recombinant murine TNF alpha injections produce a short-lived (3-6 h) dose-dependent (50-500 ng) increase in thermal and mechanical sensitivity which was significantly attenuated by prior administration of anti-NGF antiserum. 6. Intraplantar TNF alpha (100-500 ng) also elevated at 6 but not 48 h the levels of IL-1 beta and NGF in the hindpaw. 7. A single injection of anti-TNF alpha antiserum, 1 h before the CFA, at a dose sufficient to reduce the effects of a 100 ng intraplantar injection of TNF alpha, significantly delayed the onset of the resultant inflammatory hyperalgesia and reduced IL-1 beta but not NGF levels measured at 24 h. 8. The elevation of TNF alpha in inflammation, by virtue of its capacity to induce IL-1 beta and NGF, may contribute to the initiation of inflammatory hyperalgesia.

Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia.

1. Peripheral inflammation is associated with the local production of neuroactive inflammatory cytokines and growth factors. These may contribute to inflammatory pain and hyperalgesia by directly or indirectly altering the function or chemical phenotype of responsive primary sensory neurones. 2. To investigate this, inflammation was produced by the intraplantar injection of complete Freund's adjuvant (CFA) in adult rats. This resulted in a significant elevation in interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) levels in the inflamed tissue and of the peptides, substance P and calcitonin gene-related peptide (CGRP) in the L4 dorsal root ganglion 48 h post CFA injection. 3. The effects of a steroidal (dexamethasone) and a non-steroidal (indomethacin) anti-inflammatory drug on the levels of NGF and IL-1 beta in inflamed tissue were investigated and compared with alterations in behavioural hyperalgesia and neuropeptide expression in sensory neurones. 4. Systemic dexamethasone (120 micrograms kg-1 per day starting the day before the CFA injection) had no effect on the inflammatory hyperalgesia. When the dose was administered 3 times daily, a reduction in mechanical and to a lesser extent thermal sensitivity occurred. Indomethacin at 2 mg kg-1 daily (i.p.) had no effect on the hyperalgesia and a dose of 4 mg kg-1 daily was required to reduce significantly mechanical and thermal hypersensitivity. 5. The increase in NGF produced by the CFA inflammation was prevented by both dexamethasone and indomethacin, but only at the higher dose levels. Dexamethasone at the lower and higher dose regimes diminished the upregulation of IL-1 beta whereas indomethacin had an effect only at the higher dose. 6. The increase in SP and CGRP levels produced by the CFA inflammation was prevented by dexamethasone and indomethacin at the lower and higher dose regimes. 7. Intraplantar injections of IL-1 beta (0.01, 0.1 and 1 ng) produced a brief (6 h) thermal hyperalgesia and an elevation in cutaneous NGF levels which was prevented by pretreatment with human recombinant IL-1 receptor antagonist (IL-1 ra) (0.625 microgram, i.v.). The thermal hyperalgesia but not the NGF elevation produced by intraplantar IL-1 beta (1 ng) was prevented by administration of a polyclonal neutralizing anti-NGF serum. 8. IL-1 ra significantly reduced the mechanical hyperalgesia produced by CFA for 6 h after administration as well as the CFA-induced elevation in NGF levels. Anti-NGF pretreatment substantially reduced CFA-induced mechanical and thermal hyperalgesia without reducing the elevation in IL-1 beta. 9. Intraplantar NGF (0.02, 0.2 and 2 microg) injections produced a short lasting thermal and mechanical hyperalgesia but did not change IL-1beta levels in the hindpaw skin.10. Our results demonstrate that IL-1beta contributes to the upregulation of NGF during inflammation and that NGF has a major role in the production of inflammatory pain hypersensitivity.

The biphasic immunoregulation of pyrimidylpiperazine (Y-40138) is IL-10 sensitive and requires NF-kappa B targeting in the alveolar epithelium.

1. Pyrimidylpiperazine (Y-40138), a synthetic derivative of N-[1-(4-([4-(pyrimidin-2-yl)piperazin-1-yl]methyl)phenyl)cyclopropyl] acetamide, is a novel dual regulator of pro- and anti-inflammatory cytokines in vivo. The aim of the present study was to determine the signal transduction mechanisms implicated in vitro. 2. In alveolar epithelial cells, pre-treatment (30 min) with Y-40138 reduced LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha, an effect paralleled by up-regulating an anti-inflammatory counter-loop mediated through IL-10. 3. This differential regulation of pro- and anti-inflammatory signals was accompanied by an inhibition of the nuclear localization of selective NF-kappa B subunits, particularly NF-kappa B(1) (p50), RelA (p65), the major transactivating member of the Rel family, RelB (p68) and c-Rel (p75). In addition, Y-40138 blockaded, in a dose-dependent manner, the LPS-induced nuclear activation of NF-kappa B. 4. Analysis of the upstream pathway involved in Y-40138-dependent retardation of LPS-induced NF-kappa B translocation/activation revealed the involvement of an I kappa B-alpha sensitive pathway. Pre-treatment with Y-40138 ameliorated LPS-induced degradation of I kappa B-alpha in the cytosolic compartment and retarded its phosphorylation, suggesting the involvement of an upstream kinase. 5. Recombinant IL-10 (0 -- 10 ng ml(-1)) blockaded, in a dose-dependent manner, LPS-induced biosynthesis of IL-1 beta, IL-6 and TNF-alpha. Furthermore, rhIL-10 reduced the DNA binding activity of NF-kappa B. Immunoneutralization of endogenous IL-10 by a polyclonal alpha IL-10 (5 microg ml(-1)) reversed the inhibitory effect of Y-40138 on pro-inflammatory cytokines and partially restored the DNA binding activity of NF-kappa B. 6. These results indicate that Y-40138 mediated dual immunoregulation of pro- and anti-inflammatory cytokines is IL-10 sensitive and mediated through the I kappa B-alpha/NF-kappa B signal transduction pathway.

Involvement of interleukin-1 beta, nerve growth factor and prostaglandin E2 in endotoxin-induced localized inflammatory hyperalgesia.

1. Intraplantar endotoxin (ET) injection (1.25 micrograms) into the hind paw of rats resulted in a localized inflammatory hyperalgesia, as assessed by paw pressure (PP), paw immersion (PI), tail flick (TF) and hot plate (HP) tests. 2. ET injection resulted in a significant elevation in the levels of interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) in the injected foot as compared with the non-injected foot. This increase was attenuated by intraperitoneal injections of dexamethasone (200 and 400 micrograms kg-1) and to a lesser extent by indomethacin (2 and 8 mg kg-1). 3. The tripeptide Lys-D-Pro-Val, which is known to antagonize IL-1 beta and prostaglandin E2 (PGE2) reversed mechanical hyperalgesia, as assessed by the PP test, and reduced significantly thermal hyperalgesia, as assessed by the HP and TF tests. 4. IL-1ra reversed both mechanical (PP) and thermal (PI) nociceptive thresholds tested on the injected leg and significantly reduced thermal hyperalgesia, as assessed by the HP and TF tests. 5. A sheep, anti-mouse NGF antiserum reversed mechanical hyperalgesia (PP test) but had little or no effect on thermal hyperalgesia (PI, HP and TF tests). 6. Our results indicate the importance of IL-1 beta, NGF and prostaglandin E2 (PGE2) in the development of ET induced hyperalgesia and the possible existence of different mechanisms underlying thermal and mechanical as well as central and peripheral hyperalgesia.

Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13.

1. Exposure to midrange ultraviolet radiation (UVB) is known to produce skin inflammation similar to sunburn. The aim of this study was to characterize the hyperalgesia and cytokine upregulation induced by UVB and their modulation by antiinflammatory cytokines. 2. Acute exposure of the dorsal skin of mice to UVB (200, 250 and 300 mJ cm(2)) resulted in a dose-dependent decrease in the latencies of the hot plate and tail flick tests, without evident signs of skin lesions. 3. The observed hyperalgesia displayed a biphasic temporal evolution with an acute phase (3 - 6 h) and a late (48 - 96 h) phase. 4. Exposure to UVB (300 mJ cm(2)) elicited significant upregulation of interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha and nerve growth factor (NGF), determined by ELISA in the exposed skin. This upregulation was more important during the acute phase of hyperalgesia. 5. Daily treatment of mice, with i.p. injections of either IL-10 or IL-13 (1.5, 7.5 and 15 ng in 100 microl saline) produced a dose-dependent attenuation of the UVB-induced hyperalgesia. 6. Treatment with the highest doses of either IL-10 or IL-13, produced significant attenuation of the levels of the cytokines and NGF by UVB, with relatively more pronounced effects by IL-13. 7. Acute exposure to moderate amounts of UVB results in a systemic hyperalgesia related to the upregulation of cytokine and NGF levels, since both were prevented by treatment with antiinflammatory cytokines.

Nerve growth factor levels in developing rat skin: upregulation following skin wounding.

Levels of nerve growth factor (NGF) in rat hindpaw skin, measured with a sensitive two-site enzyme-linked immunosorbent assay, show two peaks during normal development. The first (57 +/- 5 pg mg-1) occurs at embryonic days (E) 18-20 and coincides with the arrival of axon terminals into the hindpaw skin. The second, larger peak (132 +/- 10 pg mg-1), occurs later, around postnatal day (P) 21 and may be involved in maintenance of neuronal phenotype. Levels outside the two peaks stay relatively constant throughout development (30 pg mg-1). Skin wounding at birth produces a marked increase in NGF levels (149 +/- 25 pg mg-1) which declines after 4 days. This large increase is not observed if wounding is performed at older ages and may underlie the sensory hyperinnervation that accompanies neonatal wounds.

Thymulin reduces hyperalgesia induced by peripheral endotoxin injection in rats and mice.

In a new model of peripheral localized inflammation, induced by intraplantar endotoxin (1.25 micrograms) injection in the hind paw of rats and mice, thymulin, a hormone of the thymus gland involved in immunomodulation, reduced inflammatory pain. High doses of thymulin reduced significantly, and in a dose-dependent manner, mechanical hyperalgesia as assessed by the paw pressure test and thermal hyperalgesia as assessed by the hot plate test and tail immersion test.

Fos-like immunoreactivity induced by intraplantar injection of endotoxin and its reduction by morphine.

C-Fos-like immunoreactivity (FLI) in the central nervous system, has been associated with the processing of nociceptive information in acute and chronic pain animal models. The aim of this study was to investigate whether intraplantar (i.pl.) injections of endotoxin (ET, 1.25 microg) can induce FLI in the lumbar spinal cord of rats and to assess the effects of morphine injection on c-fos expression. FLI was studied in various groups of rats at 2, 3, 4, 6, 9 and 24 h following ET injections. Labeled neurons were mainly detected in the lumbar segments ipsilateral to the ET-injected leg, with a major peak (71.01 +/- 4.79 positive neurons) at 4 h and a second peak (29.87 +/- 5.97 positive neurons) at 9 h followed by a recovery to the baseline at 24 h after ET injections. Within the laminae, the majority of positive neurons was observed at 2-3 h in laminae I and II and in deep laminae (V and VI mainly) starting at 4 h after ET injections. Rostrocaudally, labeled neurons were observed initially in L4-L5 segments (2-3 h post-ET) after which they extended to L2-L6 segments at 4 h after ET. Morphine injections either i.p. (1 or 2 mg/kg) or i.pl. (50 microg) significantly reduced ET-induced hyperalgesia and simultaneously the FLI. The maximum effect was observed on labeled neurons in the deep laminae (V and VI mainly). We conclude that local injections of ET can induce FLI in the lumbar spinal cord with a temporal and spatial patterns comparable to the described hyperalgesia, and that both FLI and hyperalgesia are reduced by morphine in a dose-dependent manner with a maximal effect shown by the local i.pl. morphine injections.