The role of the dorsolateral funiculi in the pain relieving effect of spinal cord stimulation: a study in a rat model of neuropathic pain.

Activation of the dorsal columns is relayed to supraspinal centers, involved in pain modulation, probably via the descending fibers in the dorsolateral funiculi (DLF). The present study examines the role of the DLF in the attenuation of pain-related signs by spinal cord stimulation (SCS). Several groups of rats were subjected to nerve injury and to chronic bilateral DLF lesions at C5-7 level. In each animal, two sets of miniature electrodes were implanted, a caudal system placed in the dorsal epidural space at low thoracic level and another implanted over the dorsal column nuclei, rostral to the lesions. Stimulation (50 Hz, 0.2 ms; 70 % of motor threshold) was applied for 5 min via either of the electrodes. Behavioral tests were used to assess the effects of SCS on the nerve injury-induced mechanical and cold hypersensitivity and heat hyperalgesia. Prior to application of SCS, antagonists to either of GABAA or B, 5-HT1 or 1-2 or α/ß-adrenergic receptors were injected i.p. Both stimulations produced comparable decreases (80-90 % of the control) of neuropathic manifestations in rats with intact spinal cords. DLF lesions attenuated the effects of both types of stimulation by about 50 %. Pretreatment with receptor antagonists differentially counteracted the effects of rostral and caudal stimulation; the inhibition with rostral stimulation generally being more prominently influenced. These results provide further support to the notion of important involvement of brainstem pain modulating centers in the effects of SCS. A major component of the inhibitory spinal-supraspinal-spinal loop is mediated by fibers running in the DLF.

Involvement of the α7-nicotinic acetylcholine receptors in the anti-inflammatory action of the thymulin-related peptide (PAT).

BACKGROUND AND PURPOSE: Peptide analog of thymulin (PAT) has been shown to have anti-hyperalgesic and anti-inflammatory properties in animal models of inflammation. Recent reports suggest that the peripheral cholinergic system has an anti-inflammatory role mediated by α7-nicotinic acetylcholine receptor (α7-nAChR). Our aim is to investigate whether the action of PAT is mediated, via the cholinergic pathway. EXPERIMENTAL APPROACH: The anti-hyperalgesic and anti-inflammatory action of PAT was assessed in rat models of inflammatory nociceptive hyperactivity (carrageenan and endotoxin) and in a mice air-pouch model for localized inflammation, respectively; the possible attenuation of PAT's effects by pretreatment with the α7-nAchR specific antagonist methyllycaconitine citrate (MLA) was also investigated. In another series of experiments, using two electrode recordings, the effect of PAT on the α7-nAChRs, expressed in Xenopus Oocytes, was also determined. KEY RESULTS: Administration of PAT reversed inflammatory nociceptive hyperactivity and cold and tactile hyperactivity in rats. This effect was partially or totally prevented by MLA, as assessed by different behavioral pain tests. Treatment with PAT also reduced the alteration of cytokines and NGF levels by carrageenan injection in the mouse air pouch model; this effect was partially antagonized by MLA. Electrophysiological recording demonstrated that PAT significantly potentiated the α7-nAchR expressed in Xenopus Oocytes. These effects were not observed when a control peptide, with a reverse sequence (rPAT), was utilized. CONCLUSIONS AND IMPLICATIONS: The behavioral and electrophysiological observations described in this report demonstrate that PAT mediates, at least partially, its anti-inflammatory action by potentiating the α7-nAChR. These results indicate that PAT has a potential for new therapeutic applications as anti-inflammatory and analgesic agent.

Spinal segmental and supraspinal mechanisms underlying the pain-relieving effects of spinal cord stimulation: an experimental study in a rat model of neuropathy.

Spinal cord stimulation (SCS) may alleviate certain forms of neuropathic pain; its mechanisms of action are, however, not fully understood. Previous studies have mainly been focused onto segmental spinal mechanisms, though there is evidence indicating a supraspinal involvement. This study aims to evaluate the relative importance of segmental and supraspinal mechanisms related to the activation of the dorsal columns (DCs). Rats were used to induce the spared nerve injury neuropathy and simultaneously subjected to chronic bilateral DC lesions at the C6-C8 level. Two pairs of miniature electrodes were implanted in each animal, with a monopolar system placed in the dorsal epidural space at a low thoracic level (below lesion) and a bipolar system placed onto the dorsal column nuclei (above lesion). Stimulation (50 Hz, 0.2 ms, 2-4V, 5 min) was applied via either type of electrodes, and tests for sensitivity to tactile and thermal stimuli were used to assess its inhibitory effects. Various receptor antagonists {bicuculline (GABA(A)), saclofen (GABA(B)), ketanserine (5HT(2)), methysergide (5HT(1-2)), phentolamine (α-adrenergic), propranolol (ß-adrenergic), sulpiride (D(2)/D(3) dopamine) or saline were injected prior to the SCS. Rostral and caudal stimulations produced a comparable inhibition of neuropathic manifestations, and these effects were attenuated by about 50% after DC lesions. Pretreatment with the various receptor antagonists differentially influenced the effects of rostral and caudal stimulation. Our findings suggest that both supraspinal and segmental mechanisms are activated by SCS, and that in this model with DC lesions, rostral and caudal stimulations may activate different synaptic circuitries and transmitter systems.

Troubleshooting in animal models of colitis: The use of a novel electrocautery model.

INTRODUCTION: Experimental colitis induced by chemical agents leads to upregulation of inflammatory cytokines in distant unaffected small intestine and to a decrease in nutrient absorption. To preclude any possible proximal diffusion of these chemicals, we designed a novel method for ulcer induction in the colon by electrocautery. METHODS: Under light anesthesia, a colonic ulcer was induced in rats by a special electrocautery probe introduced in the descending colon through the rectum allowing the injection of a controlled electrolytic current. A direct current (3-7 mA) was delivered through the electrodes for 30s and then for another 30s after reversing the polarity of the electrodes. Then, the probe was moved for a distance of +/-0.5 cm and the current injection was repeated. Rats were sacrificed at various time intervals after ulcer induction (3-96 h). Samples from colon and jejunum were taken for histological assessment and determination, by ELISA, of the levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). In other groups of animals, jejunal amino acid absorption was determined in vivo at 24 and 48 h post electrocautery. RESULTS: A colonic ulcer persisted for 72 h after cauterization. A significant upregulation of the levels of different cytokines was observed in the colon and jejunum post cauterization and persisted for at least 48 h. In the jejunum, IL-1beta increased from 81+/-9 to 652+/-110 (p<0.01) and 243+/-47 (p<0.05) pg/mg protein at 24 and 48 h, respectively. Similarly, jejunal TNF-alpha levels increased by approximately 2 folds at 24 and 48 h post ulcer induction (p<0.05). A similar but higher increase in cytokines was observed in the colon. Jejunal alanine absorption (0.2+/-0.02 micromol/20 min/cm) decreased significantly at 24 and 48 h after colitis induction (0.12+/-0.01 and 0.14+/-0.02, respectively; p<0.01). DISCUSSION: This model may be used as an alternative or a complement to chemical models of colitis.

Reversible attenuation of neuropathic-like manifestations in rats by lesions or local blocks of the intralaminar or the medial thalamic nuclei.

BACKGROUND AND AIM: Thalamic somatosensory nuclei have been classified into medial and lateral systems based on their role in nociception. An imbalance between these two systems may result in abnormal somatic sensations and spontaneous pain. This study aims to investigate the effects of transient or permanent block of the medial and intralaminar nuclear groups on the neuropathic-like behavior in a rat model for mononeuropathy. METHODS: Neuropathy was induced on one hind paw in different groups of rats following the spared nerve injury model. When the resulting hyperalgesia and allodynia (tactile and cold) reached a maximum plateau, the rats received either chemical or electrolytic lesion or lidocaine (2%) microperfusion, placed in the various thalamic nuclear groups. RESULTS: All procedures produced transient but significant decrease of neuropathic manifestations. The magnitude and duration of decrease depended on the type and the site of the block. These effects can be ranked in increasing order as follows, electrolytic

Transient attenuation of neuropathic manifestations in rats following lesion or reversible block of the lateral thalamic somatosensory nuclei.

BACKGROUND AND AIMS: Nociceptive behavior in animal models for mononeuropathy has been shown to be altered by spinal tract lesions which suggest a possible supraspinal modulation. The thalamus constitutes a chief center for the processing of nociception. We have, therefore, investigated the effects of transient or permanent blocks of the lateral somatosensory thalamic nuclei (the ventrobasal complex) on the neuropathic manifestations in rats. METHODS: Different groups of rats (n = 5-6) were subjected to mononeuropathy, following the spared nerve injury model, known to produce sustained heat hyperalgesia and tactile and cold allodynia which peaked about 2 weeks after nerve injury. This was followed by stereotaxic placement of either electrolytic or chemical lesions or implantation of mini osmotic pump for slow release of lidocaine in the ventrobasal complex. RESULTS: Chronic electrolytic and chemical lesions or reversible block of the lateral somatosensory thalamus produced transient (1-2 weeks) attenuation of neuropathic manifestations along with a persistent decrease of the hot plate latency. The most pronounced effect was observed on heat hyperalgesia, and the least significant and short-lived effect was observed on cold allodynia. CONCLUSION: We conclude that the lateral somatosensory thalamic complex is involved in the processing of neuropathic manifestations but cannot be considered as an obligatory or exclusive relay center for the neuropathic syndromes.

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 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.

Attenuation of neuropathic manifestations by local block of the activities of the ventrolateral orbito-frontal area in the rat.

Clinical and recent imaging reports demonstrate the involvement of various cerebral prefrontal areas in the processing of pain. This has received further confirmation from animal experimentation showing an alteration of the threshold of acute nociceptive reflexes by various manipulations in the orbito-frontal cortical areas. The present study investigates the possible involvement of this area in the modulation of neuropathic manifestations in awake rats. Several groups of rats were subjected to mononeuropathy following the spared nerve injury model, known to produce evident tactile and cold allodynia and heat hyperalgesia. The activity of the ventrolateral orbital areas was selectively blocked by using either chronic or acute injection of lidocaine, electrolytic lesion, or chemical lesion with kainic acid or 6-hydroxydopamine (6-OHDA). The effects of these manipulations were compared with those following lesion of the somatic sensorimotor cortical areas. Local injection of lidocaine resulted in a reversible depression of all neuropathic manifestations while electrolytic or chemical lesions elicited transient attenuation affecting mainly the heat hyperalgesia and to a lesser extent the cold allodynia. The magnitude of the observed effects with the different procedures used can be ranked as follows: 6-OHDA

Interplay between nitric oxide and vasoactive intestinal polypeptide in inducing fluid secretion in rat jejunum.

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) interact in the regulation of neuromuscular function in the gut. They are also potent intestinal secretogogues that coexist in the enteric nervous system. The aims of this study were: (1) to investigate the interaction between NO and VIP in inducing fluid secretion in the rat jejunum, and (2) to determine whether the NO effect on intestinal fluid movement is neurally mediated. The single pass perfusion technique was used to study fluid movement in a 25 cm segment of rat jejunum in vivo. A solution containing 20 mM L-arginine, a NO precursor, was perfused into the segment. The effect of the NO synthase inhibitors (L-NAME and L-nitroindazole (L-NI)) and the VIP antagonist ([4Cl-D-Phe6,Leu17]VIP (VIPa)) on L-arginine-induced changes in fluid movement, expressed as microl min(-1) (g dry intestinal weight)(-1), was determined. In addition, the effect of neuronal blockade by tetrodotoxin (TTX) and ablation of the myenteric plexus by benzalkonium chloride (BAC) was studied. In parallel groups of rats, the effect of L-NAME and L-NI on VIP-induced intestinal fluid secretion was also examined. Basal fluid absorption in control rats was (median (interquartile range)) 65 (45-78). L-Arginine induced a significant fluid secretion (-14 (-20 to -5); P<0.01). This effect was reversed completely by L-NAME (60 (36-65); P<0.01) and L-NI (46 (39-75); P<0.01) and partially by VIPa (37 (14-47); P<0.01). TTX and BAC partially inhibited the effect of L-arginine (22 (15-32) and 15 (10-26), respectively; P<0.05). The effect of VIP on fluid movement (-23 (-26 to -14)) was partially reversed by L-NAME (24 (8.4-35.5); P<0.01) and L-NI (29 (4-44); P<0.01). The inhibition of VIP or NO synthase prevented L-arginine- and VIP-induced intestinal fluid secretion through a neural mechanism. The data suggest that NO enhances the release of VIP from nerve terminals and vice versa. Subsequently, each potentiates the other's effect in inducing intestinal fluid secretion.

A thymulin analogue peptide with powerful inhibitory effects on pain of neurogenic origin.

The effects of a synthetic peptide analog of thymulin (PAT) were tested on nociceptive behavior in two animal models for peripheral mononeuropathy and in another two models for capsaicin-induced hyperalgesia. Treatment with PAT (0.25-25 microg/rat, i.p.) produced significant reduction of the mechanical allodynia and heat hyperalgesia in rats subjected to either chronic constriction injury (CCI) or spared nerve injury (SNI) models for mononeuropathy. Cold allodynia was moderately reduced in the CCI model. The inhibition of neuropathic manifestations peaked at 1-2 h post-treatment and disappeared in 3-4 h. Daily treatment with PAT, however, produced progressive attenuation of all neuropathic manifestations in the SNI model. On the other hand, pretreatment with similar doses of PAT produced dose-dependent reduction of the hyperalgesia induced by intraplantar injection of capsaicin (10 microg in 50 microl). The highest dose of PAT (50 microg) produced significant reduction of abdominal aversive behavior induced by i.p injection of capsaicin (20 microg in 100 microl). Compared with the effects of treatment with morphine or meloxicam (injected at single doses known to produce analgesia), PAT exerted equal or stronger inhibitory effects on neuropathic manifestations. The reported results suggest a possible direct action of PAT on afferent nerve fibers but its mechanisms remain to be determined.

The role of the dorsal columns in neuropathic behavior: evidence for plasticity and non-specificity.

Despite conflicting clinical and experimental evidence, textbook description of somatic sensations continues to follow a rigid dichotomy based on the concept that pain sensation is transmitted cephalad primarily through anterolateral pathways, while touch is mediated through the dorsal column pathway. This study provides an example of the dynamic rerouting in the transmission of the nociceptive signals following injuries to the peripheral and central processes of sensory neurons. In two rat models for mononeuropathy, the chronic constriction injury model [Bennett, G.J., Xie, Y.K., Pain 33 (1988) 87-107] and the spared nerve injury model [Decosterd, I., Woolf, C.J., Pain 87 (2000) 149-158], we demonstrate that selective dorsal columns lesion produced significant decrease of tactile and cold allodynias and thermal hyperalgesia which were assessed by the Von Frey hair filaments, the acetone drop test and the heat-induced paw withdrawal, respectively. These manifestations, however, can reappear 2 weeks after bilateral dorsal column lesion in rats subjected to spared nerve injury mononeuropathy and appear also in animals sustaining chronic bilateral dorsal column lesion followed by either model of mononeuropathy. Lesion of the dorsal column on the side opposite to the neuropathic leg did not alter the neuropathic manifestations in both animal models. Changes in the sequence of timing of the dorsal column lesion and induction of mononeuropathy, suggest that the effects of the former last for 1 to 2 weeks. The results of this study show that the dorsal columns are involved in neuropathic manifestations and at the same time are not necessary for their full development and persistence. Furthermore, these results shade doubts on the validity of the concept of segregation of pathways involved in the transmission of neuropathic manifestations. Therefore, principles governing acute pain transmission are not necessarily applicable to chronic pain situations. The latter conditions seem to engage other available pathways to reestablish the pain signaling system.

Upregulation of proinflammatory cytokines and nerve growth factor by intraplantar injection of capsaicin in rats.

Capsaicin-sensitive primary afferents (CSPA) are known to be involved in nociception and neurogenic inflammation. Extensive research has been devoted to the sensory role of these fibres but less attention has been paid to their local effector function. This study aimed at gaining more insight into the molecular mechanisms underlying the neurogenic inflammation induced by this special group of afferent fibres. Different groups of rats (n = 5 in each group), either naive or subjected to selective ablation of their CSPA, received individual intraplantar injections of saline, capsaicin, its vehicle or capsaicin preceded by its antagonist, capsazepine. Acute tests for nociception were used to assess the variations of the nociceptive thresholds. Variations of the levels of proinflammatory cytokines and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Intraplantar injection of capsaicin (10 microg in 50 microl) produced a sustained thermal and mechanical hyperalgesia that peaked at 3-6 h and disappeared 24 h following the injection. Similar capsaicin injection in further groups of rats produced an early upregulation of the proinflammatory cytokines and NGF, which peaked at 30-60 min and returned to control levels within 2-5 h. Similar effects were observed following the application of either capsaicin or intense electrical stimulation on the cut end of the distal portion of the sciatic nerve. The effects of capsaicin were abolished in rats subjected to selective ablation of their CSPA. These results demonstrate that CSPA can simultaneously challenge the immune system through the release of proinflammatory mediators and the central nervous system through nociceptive signalling and can therefore serve as a common afferent pathway to both immune and nervous systems.

Attenuation of neuropathic pain by segmental and supraspinal activation of the dorsal column system in awake rats.

In addition to its involvement in the transmission of neuropathic pain, the dorsal column system has been shown to have analgesic effects when electrically stimulated. The segmental or supraspinal origin of the analgesia, however, has not been clearly delineated. The aim of this study is to demonstrate the contribution of supraspinal mechanisms to the inhibition of allodynia and hyperalgesia in two different rat models of mononeuropathy. Mononeuropathy was induced, under deep anesthesia, in several groups of rats (n=7 each) following either the chronic constriction injury or the spared nerve injury model. Mechanical and cold allodynia were assessed by the Von Frey monofilaments and by the acetone drop test, respectively. Thermal hyperalgesia was assessed by the paw withdrawal and hot plate tests. Bipolar electrodes for dorsal column stimulation were implanted chronically in all rats on the dorsal aspect of the medulla at the level of the obex. Selective dorsal column bilateral lesions were performed at the upper cervical level in some groups of rats. Dorsal column nuclear stimulation, rostral to selective dorsal spinal lesions, produced strong inhibitory effects on the allodynia and hyperalgesia observed in both models of mononeuropathy. These effects were comparable to those observed following similar stimulations in rats with an intact spinal cord. Our results demonstrate strong inhibitory effects of dorsal column stimulation on neuropathic pain. This inhibition can be attributed to the activation of brainstem pain-modulating centers via rostral projections of the dorsal column nuclei.

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 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.

Alpha-melanocyte-related tripeptide, Lys-d-Pro-Val, ameliorates endotoxin-induced nuclear factor kappaB translocation and activation: evidence for involvement of an interleukin-1beta193-195 receptor antagonism in the alveolar epithelium.

The potential anti-inflammatory role of alpha-melanocyte-stimulating hormone (alpha-MSH)-related tripeptide, lysine(11)-D-proline-valine(13) (KDPV), an analogue of interleukin (IL)-1beta(193-195) and an antagonist of IL-1beta/prostaglandin E(2), is not well characterized in the alveolar epithelium. In a model of foetal alveolar type II epithelial cells in vitro, we showed that lipopolysaccharide endotoxin (LPS) differentially, but selectively, induced the nuclear subunit composition of nuclear factor kappaB(1) (NF-kappaB(1)) (p50), RelA (p65) and c-Rel (p75), in parallel to up-regulating the DNA-binding activity (supershift indicating the presence of the p50-p65 complex). LPS accelerated the degradation of inhibitory kappaB-alpha (IkappaB-alpha), accompanied by enhancing its phosphorylation in the cytosolic compartment but not in the nucleus. KDPV suppressed, in a dose-dependent manner, the nuclear localization of p50, p65 and p75, an effect that led to the subsequent inhibition of NF-kappaB activation. Interleukin-1 receptor antagonist (IL-1ra) decreased the nuclear abundance of p50, p65 and p75, and subsequently depressed the DNA-binding activity induced by LPS. Analysis of the mechanism involved in the KDPV- and IL-1ra-mediated inhibition of NF-kappaB nuclear localization revealed a reversal in IkappaB-alpha phosphorylation and degradation, followed by cytosolic accumulation. LPS induced endogenous IL-1beta biosynthesis in a time-dependent manner; the administration of exogenous recombinant human interleukin 1 (rhIL-1) resulted in a dose-dependent activation of NF-kappaB. KDPV and IL-1ra abrogated the effect of rhIL-1. Pretreatment with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, an inhibitor of cyclo-oxygenase, blocked the LPS-induced activation of NF-kappaB. These results indicate the involvement of prostanoid-dependent (NSAID-sensitive) and IL-1-dependent (IL-1ra-sensitive) mechanisms mediating LPS-induced NF-kappaB translocation and activation, a pathway that is regulated, in part, by a negative feedback mechanism transduced through IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB.

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.

Thiol regulation of pro-inflammatory cytokines reveals a novel immunopharmacological potential of glutathione in the alveolar epithelium.

The therapeutic immunopharmacological potential of glutathione in the alveolar epithelium is not well characterized. We developed an in vitro model of fetal alveolar type II epithelial cells to investigate the effect of redox disequilibrium on chemioxyexcitation (DeltapO(2)/ROS) induced up-regulation of pro-inflammatory cytokines. Buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione (GSH) biosynthesis, induced intracellular reactive oxygen species (ROS) and the release of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha. Chloroethyl nitrosourea, which blocks the NADPH-dependent recycling of oxidized glutathione (GSSG), reduced ROS-induced cytokine production, similar to pyrrolidine dithiocarbamate, an antioxidant/pro-oxidant thiuram, which elevates GSSG. The antioxidant and GSH precursor, acetylcysteine, abrogated cytokine release concomitant with suppression of ROS, an effect mimicked by gamma-glutamylcysteinyl-ethyl ester, a cell permeant GSH. Cysteine, the rate-limiting amino acid in the de novo synthesis of GSH, administered as oxothiazolidine carboxylate and adenosylmethionine, mitigated the chemioxyexcitation-dependent cytokine release. Ebselen, an anti-inflammatory antioxidant, which mimics the effect of glutathione peroxidase, neutralized ROS by the GSH-peroxidase-coupled reaction, thereby blocking the pathway to cytokine enhancement. Our results indicate that modulating redox equilibrium by pharmacological thiols exhibits differential regulation on pro-inflammatory cytokines, thus bearing clinical consequences for the therapeutic treatment of pediatric distresses in pathophysiology.

A perfusion technique for the determination of pro-inflammatory mediators induced by intradental application of irritants.

INTRODUCTION: Several morphological and functional features contribute to the consideration of the tooth as a separate compartment having special type of innervation and special immune mechanisms. This study describes a new method allowing the intradental perfusion of rat incisors for the in vivo assessment of pulpal reaction to inflammatory agents. METHODS: Under deep anesthesia, the distal 2-3 mm of each of the rat lower incisors was cut and wrapped in a polyethylene tubing connected to a perfusion chamber made of tigone tubing (ID 1/8 in., volume 100-150 microl). Several groups of rats (n=5 each) were used for intradental application of either saline, capsaicin (100 microg in 100 microl), or endotoxin (ET, 20 microg in 100 microl) for a period of 40 min followed by filling the tooth chamber with saline and collecting the perfusate every 40 min for a period of 8 h. The collected perfusates were stored at -70 degrees C for subsequent determination of the concentration of prostaglandin E(2) (PGE(2)) and nerve growth factor (NGF) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Dentinal injury produced a moderate increase in the levels of NGF and PGE(2) in incisors perfused with saline. Application of ET or capsaicin, however, produced a highly significant increase in the levels of both mediators. These effects peaked at 1.5-3 h for PGE(2) and at 5 h for NGF. Capsaicin showed the most significant effects. DISCUSSION: The reported results cannot be attributed to any factor other than the inflammation of the incisor's pulp, because the described chamber does not allow any spread or leak of the applied irritants. Further studies using other reagents can allow the determination of the variation of the levels of the various pro-inflammatory mediators and their modulation by treatment with anti-inflammatory drugs.