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.

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.

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 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.

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.

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.

Gangrenous cholecystitis: analysis of risk factors and experience with laparoscopic cholecystectomy.

BACKGROUND: Gangrenous cholecystitis occurs in up to 30% of patients admitted with acute cholecystitis. Factors predicting gangrenous disease in patients with acute cholecystitis remain poorly defined, making preoperative diagnosis difficult. Identification of these factors and early diagnosis of gangrenous cholecystitis will indicate more aggressive treatment, earlier operation, and a lower threshold for conversion of laparoscopic to open cholecystectomy. METHODS: We reviewed our experience with acute cholecystitis during the 2-year period of 1995 to 1996. Admitting history, physical examination, operative report, laboratory and radiology data, and pathology report were analyzed for each patient. Acute cholecystitis and its gangrenous complication were diagnosed by both gross and microscopic examination. RESULTS: One hundred fifty-four patients were admitted to the hospital with acute cholecystitis and underwent cholecystectomy; gallbladder gangrene was found in 27 (18%) of these patients. Four patients with gallbladder gangrene underwent open cholecystectomy and 23 patients underwent laparoscopic cholecystectomy, of which 15 (65%) were completed laparoscopically and 8 (35%) had open conversion as a result of severe inflammation. Risk factors for gallbladder gangrene included male gender, age older than 50 years, history of cardiovascular disease, and leukocytosis greater than 17,000 white blood cells/mL. CONCLUSIONS: Older male patients (age older than 50 years) with history of cardiovascular disease, leukocytosis greater than 17,000 white blood cells/mL, and acute cholecystitis have increased risk of gallbladder gangrene and conversion of laparoscopic cholecystectomy to open cholecystectomy. Urgent laparoscopic cholecystectomy with low threshold for conversion to open cholecystectomy should be considered in these patients at high risk for gallbladder gangrene.

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.

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.

Cytokine-mediated or direct effects of thymulin on the nervous system as assessed by pain-related behavior.

Thymulin is a thymic hormone with known immunomodulatory activities. Recent evidence has indicated a signaling role for this peptide in the interaction between the immune, endocrine and the nervous system. In this report, we review recent experimental findings on the analgesic actions of thymulin (high doses) in rats with endotoxin-induced localized inflammation and the hyperalgesic actions (low doses) of this peptide in intact animals. These actions involve both proinflammatory cytokines and PGE2. The possibility of a dual role played by thymulin as a hormone that might also involve a direct effect on the nervous system is discussed.

Involvement of interleukin-1 beta, nerve growth factor, and prostaglandin-E2 in the hyperalgesia induced by intraplantar injections of low doses of thymulin.

The effect of various doses of intraplantar thymulin injection, on nociceptive thresholds, in the hind paw of rats was assessed using different pain tests. As little as 0.5 ng thymulin resulted in localized mechanical hyperalgesia as assessed by the paw pressure test and thermal hyperalgesia as assessed by the paw immersion, hot plate, and tail flick tests. The highest dose of thymulin (10 ng) reduced both paw pressure and paw immersion latencies in the noninjected paw also. Thymulin (5 ng) also resulted in significant elevation in the levels of interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) levels in the injected paw. Both dexamethasone and indomethacin reversed thymulin-induced hyperalgesia. Also interleukin-1 receptor antagonist (IL-1ra) and a polyclonal anti-NGF antiserum significantly reduced thymulin-induced hyperalgesia. On the other hand, the tripeptide lys-D-pro-val (known to antagonize IL-1 beta and PGE2 induced hyperalgesia) reversed the hyperalgesia due to thymulin. In conclusion, thymulin induces localized hyperalgesia which is mediated by PGE2-dependent mechanisms and this pathway could be either partially dependent on or totally independent of IL-1 beta mechanisms.

Chemioxyexcitation (delta pO2/ROS)-dependent release of IL-1 beta, IL-6 and TNF-alpha: evidence of cytokines as oxygen-sensitive mediators in the alveolar epithelium.

The signalling mechanisms in oxidative stress mediated by cytokines in the perinatal alveolar epithelium are not well known. In an in vitro model of fetal alveolar type II epithelial cells, we investigated the profile of cytokines in response to ascending Deltap O(2)regimen (oxyexcitation). The peak of TNF-alpha (4 h) preceded IL-1beta and IL-6 (6-9 h), indicating a positive feedback autocrine loop confirmed by exogenous rmTNF-alpha. Reactive oxygen species (ROS) induced a dose-dependent release of cytokines, an effect specifically obliterated by selective antioxidants of the hydroxyl radical (*OH) and superoxide anion (O(2)-). Actinomycin and cycloheximide blocked the induced production of cytokines, implicating transcriptional and translational control. Whilst the dismutating enzymes superoxide dismutase (SOD) and catalase were ineffective in reducing ROS-induced cytokines, MnP, a cell-permeating SOD mimetic, abrogated xanthine/xanthine oxidase-dependent cytokine release. Desferrioxamine mesylate, which inhibits the iron-catalysed generation of *OH via the Fenton reaction, exhibited a mild effect on the release of cytokines. Dynamic variation in alveolar p O(2)constitutes a potential signalling mechanism within the perinatal lung allowing upregulation of cytokines in an ROS-dependent manner.

Effects of various analgesic and anti-inflammatory drugs on endotoxin-induced hyperalgesia in rats and mice.

A new model of endotoxin (ET)-induced hyperalgesia has been used to test the effects of four classes of drugs in rats and mice. Hyperalgesia was assessed by paw pressure (PP), hot plate (HP) and tail flick (TF) tests. Each drug was injected intraperitoneally 24 and 12 h before ET injection and just before each pain test at 3, 6, 9 and 24 h after ET injection. At the dosages used, acetaminophen and dexamethasone were the most effective in reducing PP hyperalgesia and least effective on TF hyperalgesia, while indometacin and morphine produced their main effect on TF hyperalgesia. The four drugs were about equally effective in reversing HP hyperalgesia. We conclude that ET hyperalgesia is mediated by both prostaglandin-sensitive and prostaglandin-independent mechanisms.