Transcriptional expression of inflammatory mediators in various somatosensory relay centers in the brain of rat models of peripheral mononeuropathy and local inflammation.

Contradictory results have been reported regarding the role of inflammatory mediators in the central nervous system in mediating neuropathic pain and inflammatory hyperalgesia following peripheral nerve injury or localized inflammation. The present study aims to correlate between the mRNA expression and protein secretion of proinflammatory cytokines and nerve growth factor (NGF), in the dorsal root ganglia (DRGs), spinal cord, brainstem and thalamus, and pain-related behavior in animal models of peripheral mononeuropathy and localized inflammation. Different groups of rats (n=8, each) were subjected to either lesion of the nerves of their hindpaws to induce mononeuropathy or intraplantar injection of endotoxin (ET) and were sacrificed at various time intervals. TNF-α, IL-1ß and NGF mRNA expression and protein levels in the various centers involved in processing nociceptive information were determined, by RT-PCR and ELISA. Control groups were either subjected to sham surgery or to saline injection. Mononeuropathy and ET injection produced significant and sustained increases in the mRNA expression and protein levels of TNF-α, IL-1ß and NGF in the ipsilateral and contralateral DRGs, spinal cord, and brainstem. No significant and consistent changes in the mRNA expression of cytokines were noticed in the thalamus, while a downregulation of the NGF-mRNA level was observed. The temporal and spatial patterns of the observed changes in mRNA expression of cytokines and NGF are not closely in phase with the observed allodynia and hyperalgesia in the different models, suggesting that the role of these mediators may not be reduced exclusively to the production and maintenance of pain.

Brainstem injection of lidocaine releases the descending pain-inhibitory mechanisms in a rat model of mononeuropathy.

Lidocaine injections in the rostral ventromedial medulla (RVM) have been shown to produce significant reduction of neuropathic manifestations in rats. This effect has been attributed to selective block of a pain descending facilitatory system, responsible for chronic pain. However, recent observations from our laboratory did not provide confirmation to this hypothesis. We aimed, therefore, to investigate the spinal synaptic mechanisms activated by lidocaine injections in the RVM. Rats were subjected, under deep anesthesia, to the induction of mononeuropathy on one hindpaw, and to the stereotaxic implantation of chronic cannulae in the RVM for the injection of lidocaine or GABA antagonists. Implanted intrathecal catheter in the lumbosacral space was used for the injection of specific antagonists to GABA, 5HT, glycine, noreadrenaline and dopamine, prior to lidocaine. Tactile and cold hyperreactivity and heat hyperalgesia were assessed using von Frey hair filaments, acetone drop test and heat-induced paw withdrawal, respectively. Lidocaine injections produced significant inhibition of all neuropathic manifestations. Intrathecal injection of antagonists to GABA (bicucullin, picrotoxin and saclofen), serotonin 5HT(1-2) (ketanserin and methysergide) and α- (phentoalmine, yohimbine) and ß- (propranolol) adrenergic receptors, suppressed the lidocaine inhibitory effects; while partial or no attenuation were observed following pretreatment with glycine and dopamine D(2/3) antagonists. Comparable effects were observed with RVM injection of GABA antagonists. Lidocaine injection in the RVM results in a release of the descending pain-inhibitory systems from a tonic gabaergic inhibition. This descending system involves the activation of gabaergic, serotonergic and adrenergic mechanisms at the level of the spinal dorsal horn.

Thymulin related peptide attenuates inflammation in the brain induced by intracerebroventricular endotoxin injection.

Based on significant amount of evidence, it is now generally believed, that one underlying cause for neurodegenerative diseases, could be dysregulation in inflammatory processes. The actual mechanisms involved are not yet well understood. Several studies have demonstrated the potent analgesic and anti-inflammatory actions of thymulin related peptide (PAT), in different animal pain models. In this study, we investigated the efficacy of PAT in a recently developed model of neuroinflammation, in conscious rats, caused by intracerbroventricular (ICV) injection of endotoxin (ET). Our results indicate that ICV injection of PAT alone did not elicit significant alteration of nociceptive thresholds, while ET injections produced significant thermal hyperalgesia and cold allodynia. Pretreatment with PAT resulted in significant alleviation of ET-induced hyperalgesia and increased body temperature. In other sets of experiments, ICV injection of ET resulted in a significant elevation in the concentration of pro-inflammatory mediators measured in different areas of the brain; this elevation was significantly following pretreatment with PAT. Taken together these results provide evidence in support of our hypothesis that as a potent anti-inflammatory and analgesic peptide, PAT might have potential therapeutic use for the treatment of neurodegenerative conditions induced by silent or overt inflammation.

Alteration of GABAergic and glycinergic mechanisms by lidocaine injection in the rostral ventromedial medulla of neuropathic rats.

Attenuation of neuropathic manifestations in experimental animals, by lidocaine injection in the rostral ventro-medial medulla (RVM), has been traditionally attributed to selective block of a descending pain facilitatory system. However, the presence of descending fibers carrying this effect and the selective action of lidocaine on the facilitatory neurons, have not been supported by convincing experimental evidence. The present study aimed to investigate the mechanisms underlying the hypoalgesic action of lidocaine injection in the brainstem. Several groups of rats were subjected to mononeuropathy on their left hind paws, according to the model of spared nerve injury, and were subsequently implanted with guide cannulae in the RVM. After recovery, rats received injections of lidocaine, GABA and glycine agonists or antagonists and their effects were assessed on behavioral tests of allodynia and hyperalgesia. Injections of lidocaine at doses ranging between 0.05% and 2% produced attenuation at high doses and no effects or increasing hyperalgesia at low doses. GABA and glycine agonists increased neuropathic manifestations while their antagonists elicited the opposite effects. A combined injection of GABA agonist or glycine with lidocaine (0.5%) prevented the inhibitory effects of lidocaine injection alone. Our results are in line with the abundant documentation on the alteration of the function of inhibitory neurons by lidocaine and reveal a possible action of the injected high doses on the GABAergic and glycinergic neurons in the RVM. The resulting block of the inhibitory tone exerted by these neurons can lead to a release of the descending pain inhibitory systems.

Inflammatory mediators released following application of irritants on the rat injured incisors. The effect of treatment with anti-inflammatory drugs.

The exchange of fluids and chemicals between the tooth pulp and the periphery, through the dentinal tubules has been well documented. Application of irritants on the open tubules produces aversion in awake rats that can be prevented by prior occlusion of these tubules. This study aims at characterizing the secretion of inflammatory mediators in tooth perfusates and assessing the effects of systemic pretreatment with anti-inflammatory drugs on the levels of these mediators. Several groups of rats (n=5-6 each) were used for intradental application of either saline, capsaicin (100 microg in 100 microl), or endotoxin (20 microg in 100 microl) for a period of 40 min followed by filling the perfusion chamber with sterile saline and collecting the perfusate every 30 min for 6h. The perfusates were used for the determination of the concentrations of cytokines by ELISA. Application of irritants produced a highly significant increase in PGE2 (peak at 2h) and NGF (peak at 4-6h). Dexamethasone antagonized the effects of endotoxin and capsaicin, while NSAID affected mainly the endotoxin-induced inflammation. Our results confirm the validity of the tooth perfusion technique and demonstrate that the efficacy of treatment with anti-inflammatory drugs depends on the type of inflammation.

Transient increase in cytokines and nerve growth factor in the rat dorsal root ganglia after nerve lesion and peripheral inflammation.

Inflammatory response occurs, in general, at the peripheral site of injury or irritation and in the corresponding spinal dorsal horn segments. In this study, we show transient increases in the protein concentrations of interleukins 1beta, 6, 8 and NGF in lumbar DRGs within hours after sciatic mononeuropathy in the rat. Comparable increases, with variation in temporal patterns, were observed after skin inflammation induced by intraplantar injection of endotoxin or complete Freund's adjuvant. The non-matching temporal profiles of cytokines and NGF increases and those of behavioral hypersensitivity, particularly peak values, suggest that the roles of these proteins are not necessarily pro-nociceptive in the DRGs at the chronic phase of neuropathy.

Nociceptive behavior in animal models for peripheral neuropathy: spinal and supraspinal mechanisms.

Since the initial description by Wall [Wall, P.D., 1967. The laminar organization of dorsal horn and effects of descending impulses. J. Neurophysiol. 188, 403-423] of tonic descending inhibitory control of dorsal horn neurons, several studies have aimed to characterize the role of various brain centers in the control of nociceptive input to the spinal cord. The role of brainstem centers in pain inhibition has been well documented over the past four decades. Lesion to peripheral nerves results in hypersensitivity to mild tactile or cold stimuli (allodynia) and exaggerated response to nociceptive stimuli (hyperalgesia), both considered as cardinal signs of neuropathic pain. The increased interest in animal models for peripheral neuropathy has raised several questions concerning the rostral conduction of the neuropathic manifestations and the role of supraspinal centers, especially brainstem, in the inhibitory control or in the abnormal contribution to the maintenance and facilitation of neuropathic-like behavior. This review aims to summarize the data on the ascending and descending modulation of neuropathic manifestations and discusses the recent experimental data on the role of supraspinal centers in the control of neuropathic pain. In particular, the review emphasizes the importance of the reciprocal interconnections between the analgesic areas of the brainstem and the pain-related areas of the forebrain. The latter includes the cerebral limbic areas, the prefrontal cortex, the intralaminar thalamus and the hypothalamus and play a critical role in the control of pain considered as part of an integrated behavior related to emotions and various homeostatic regulations. We finally speculate that neuropathic pain, like extrapyramidal motor syndromes, reflects a disorder in the processing of somatosensory information.

Ultra violet-induced localized inflammatory hyperalgesia in awake rats and the role of sensory and sympathetic innervation of the skin.

Exposure to mid range ultrat violet radiations (UVBs) has been shown to produce systemic inflammation and hyperalgesia in mice [Saadé, N.E., Nasr, I.W., Massaad, C.A., Safieh-Garabedian, B., Jabbur, S.J., Kanaan, S.A., 2000. Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13. Br. J. Pharmacol. 131, 1317-1324]. Our aim was to characterize a new rat model of localized exposure to UVB and to determine the role of skin innervation in the observed hyperalgesia and cytokine upregulation. In several groups of rats one hindpaw was exposed to UVB (250-350 mJ/cm(2)) and this was followed by the application, to the plantar area of the paw, of either Von Frey hairs or a few acetone drops to measure tactile and cold allodynia, respectively. Thermal hyperalgesia was assessed by the paw withdrawal latency and duration. Cytokine levels were determined, by ELISA, in processed samples of skin tissue isolated from the exposed and non-exposed paws. UVB induced a biphasic thermal hyperalgesia and cold and tactile allodynia with an early phase that peaked at 3-6h and disappeared at 24h and a late phase with a peak at 48 h and recovery at 72-h post-exposure. Tumor necrosis factor, interleukins 1 beta, 6, 8, 10 and NGF levels were significantly increased following the same biphasic temporal pattern. Chemical ablation of capsaicin sensitive afferents and guanethidine injection produced significant alteration of the hyperalgesia and allodynia. The increase in cytokine levels by UVB was also altered by both treatments. The present study describes a new animal model for localized UVB-induced inflammatory hyperalgesia and provides evidence about the involvement of neurogenic mechanisms in the observed hyperalgesia and upregulation of proinflammatory mediators.

Spinal pathways involved in supraspinal modulation of neuropathic manifestations in rats.

Controversial results have been recently reported on the role of supraspinal centers in the modulation of nociceptive behavior in animal models of mononeuropathy. Our aim was to investigate the role of the various spinal pathways in the modulation of the neuropathic manifestations. Several groups of rats were subjected to selective spinal-tract lesions, either 2-3 weeks before or 2-3 weeks after the induction of mononeuropathy following the chronic constriction injury (CCI) or the spared nerve injury (SNI) models. Tactile and cold allodynias were assessed by Von Frey filaments and the acetone drops test, respectively. Thermal hyperalgesia was assessed by the paw withdrawal and the hot plate tests. The effects of unilateral and bilateral lesions of the dorso-lateral funiculus (DLF), the anterolateral column (ALC) or hemisection were tested over a period of 4-8 weeks. All spinal tract lesions produced reversible, but significant decrease of allodynia and hyperalgesia over a period of 1-3 weeks. The most pronounced effects were observed with bilateral lesions. The stronger attenuation was observed on thermal hyperalgesia, assessed by the paw withdrawal test, while cold allodynia was the least affected. Spinal lesions performed before the induction of neuropathy did not produce significant alterations in the temporal development of neuropathic manifestations. The present results allow the conclusion that all spinal tracts can be involved in the rostral transmission and the descending modulation of neuropathic manifestations. The recovery of symptoms following spinal lesions provides illustration on the plasticity of the neural network involved in the processing of the neuropathic syndromes.

Effects of ventral hippocampal lesion on thermal and mechanical nociception in neonates and adult rats.

The proper maturation of the hippocampus is essential for the development of different behaviours, including memory, pain responses and avoidance. The mechanisms involved in the neurodevelopment of nociception have also been implicated in several neuropsychiatric disorders. The neonatal lesion of the ventral hippocampus (VH) in rats, an animal model of schizophrenia, can be utilized to study the developmental neurobiology of animal behaviour. We examined the nociceptive responses in this animal model at different stages of development. Rat pups were lesioned at postnatal day 7 by injecting ibotenic acid into the VH bilaterally, and then tested for thermal and mechanical nociception at the age of 35, 65 and 180 days. The nociceptive tests used were the hot plate (HP), paw pressure (PP) and tail flick (TF) tests. Another group of adult rats had the same lesion in the VH and then underwent the same tests at 28, 56 and 168 days post-lesions. When compared with sham controls, the rats with neonatal VH lesion showed decreased latency for the HP and PP tests only after puberty. The TF test showed significant increase in latency for both groups at age 65 and 180 days. The adult rats with VH lesion showed no major changes over all periods of testing. These results suggest that early lesion of VH can alter the development of the neural mechanisms involved in the processing of thermal and mechanical nociception.

Continuous perfusion with morphine of the orbitofrontal cortex reduces allodynia and hyperalgesia in a rat model for mononeuropathy.

Recent imaging reports demonstrate the activation of the orbitofrontal cortical (OFC) area during acute and chronic pain. The aim of this study was to compare the effects of chronic perfusion of this area with morphine on nociception in control rats and in rats subjected to mononeuropathy. Chronic perfusion of morphine, using miniosmotic pumps, produced significant and naloxone-reversible depression of tactile and cold allodynias and thermal hyperalgesia, observed in neuropathic rats, while it produced significant elevation and naloxone insensitive increase of acute nociceptive thresholds in control rats. The observed results support the idea that this area is a component of a flexible cerebral network involved in pain processing and perception.

Occlusion of dentinal tubules and selective block of pulp innervation prevent the nociceptive behaviour induced in rats by intradental application of irritants.

OBJECTIVES: Application of irritants on the exposed dentine of the incisors has been shown to produce aversive behaviour in awake rats. This study aims to demonstrate that the observed aversion is due to the infiltration of irritants through the dentinal tubules and the activation of capsaicin sensitive fibres in the tooth pulp. METHODS: Different groups of rats were subjected, under anaesthesia, to cutting of the distal 2 mm of their lower incisors and the fixation of an artificial crown that allows the application of 10-15 microl of solution. Several procedures were followed to prevent the action of the irritants including occlusion of the dentinal tubules, local application of lidocaine, selective ablation of the capsaicin sensitive primary afferents (CSPA) or incisor pulpectomy; the reactions to intradental application of either capsaicin (1%) or formalin (2.5%) were tested using a newly designed behavioural score. RESULTS: Occlusion of dentinal tubules produced significant attenuation of the nociceptive behaviour induced by dentinal application of either capsaicin or formalin. Similar results were observed following either local block with lidocaine (2%), selective ablation of capsaicin sensitive afferents or total denervation by pulpectomy. CONCLUSIONS: The present results confirm the hypothesis of infiltration of irritants to the incisor pulp through the dentinal tubules and suggest that the reported inflammatory reaction and hyperalgesia are mediated, to a large extent, by capsaicin sensitive primary afferents.

Thymulin reduces the hyperalgesia and cytokine upregulation induced by cutaneous leishmaniasis in mice.

Cutaneous leishmaniasis (CL) in mice has been shown to produce hyperalgesia and upregulation of interleukin (IL)-1beta and nerve growth factor (NGF) levels. The aim of this study was to investigate the effects of thymulin on CL-induced hyperalgesia and cytokine upregulation. Daily treatment with thymulin (1, 100, and 1000 ng/ip) produced dose-dependent decreases in CL-induced hyperalgesia as assessed by the tail flick and the hot plate tests. The levels of NGF and IL-1beta were determined in the skin tissues of the hind leg in different groups (n = 5 each) of mice over a period of 5 weeks. Mice with CL showed sustained increase in the levels of IL-1beta and NGF which were reversed by thymulin (1 microg). Injection of thymulin only did not alter the nociceptive thresholds or the levels of IL-1beta and NGF. We conclude that thymulin can modulate the hyperalgesia induced by CL by decreasing the levels of the proinflammatory factors IL-1beta and NGF.

Nociceptive behaviour induced by dental application of irritants to rat incisors: a new model for tooth inflammatory pain.

Animal models simulating acute human pulpitis are still lacking. The rat incisors present a particular situation where most of their innervation is considered to be unmyelinated and concentrated mainly in the tooth pulp. This study reports on a new model for dental pain induced by inflammatory agents applied to the tooth pulps of incisors. In different groups of rats, artificial crowns were fixed on the lower incisors, after cutting 1-2mm of their distal extremities. A volume of 7-10 microl of solutions of saline, capsaicin (1-10mg/ml) or formalin (2.5% or 5%) was injected in the crown cavity, and the nociceptive behaviour was quantitated following a devised scoring method of four scales. Intradental application of capsaicin produced nociceptive scores in the form of one plateau for 1-2h depending on the concentration used. Similar results were obtained with intradental application of formalin 2.5%. The one plateau of nociceptive scores obtained with formalin contrasts with the biphasic aspect of nociceptive behaviour described with the intradermal formalin test. This discrepancy could be attributed to a difference in the types of afferent fibres involved in each situation. Pretreatment with morphine (2 mg/kg) attenuated, in a naloxone-reversible manner, the nociceptive behaviour observed following intradental application of capsaicin. Pretreatment with meloxicam (a cyclo-oxygenase-2 inhibitor) exerted a less pronounced attenuation of the nociceptive scores when compared with morphine. These results provide evidence for the validity of the described model for the simulation of tooth pulp inflammatory pain in awake animals.

Hyperalgesia and upregulation of cytokines and nerve growth factor by cutaneous leishmaniasis in mice.

Classical description of syndromes produced by cutaneous leishmaniasis (CL) does not include sensory manifestations such as pain and/or itching, despite the evident upregulation of proinflammatory cytokines. Using a murine model of CL we report on evident hyperalgesia, as assessed by acute pain tests, and sustained upregulation of interleukin (IL-1beta) and nerve growth factor (NGF). This upregulation, especially that of NGF, may explain the observed hyperalgesia, in the light of recent evidence on the role of cytokines in the sensitization of nerve afferents and the subsequent hyperalgesia.

Chronic thalamotomy increases pain-related behavior in rats.

The thalamus has been traditionally considered as the 'chief organ' by which pain is perceived (Head H, Holmes G. Sensory disturbances from cerebral lesions. Brain 1911;34:102-254). However, several clinical and experimental observations led to a challenge of this traditional view. In this report, we demonstrate that chronic thalamic lesions, instead of producing hypoalgesia, increased pain reactivity in rats. Different groups of rats were subjected to either subtotal, lateral or medial thalamic lesions. Their reactions to nociceptive stimuli were then assessed for a period of 1-2 months. Rats in the different groups showed an increased reactivity to acute mechanical and thermal nociceptive stimuli and an increase in the pain scores of the formalin test. These results suggest an important role of the thalamus in pain modulation in addition to that of nociceptive transmission.

From electrical wiring to plastic neurons: evolving approaches to the study of pain.

Clinical and animal experimental evidences accumulated during the past four decades indicate an evolving change, championed by Patrick D. Wall, from the old concept of a specific pain pathway and hard-wired nervous system to a dynamic concept of plastic neural mechanisms underlying nociceptive processing like other sensory neural functions. These include: (1) the reciprocal sharing and interaction of various somatic sensory modalities between the ascending pathways; (2) the activation of spinal gating mechanisms through a dorsal column brainstem spinal loop; (3) the role of plastic changes in the nervous system in the production and maintenance of chronic pain; (4) the evidence showing that processing of nociception involves the activation of a diffuse network of transmitting fiber tracts and brain centers that are not exclusively devoted to pain; and (5) the consideration of chronic pain, at least in part, as a sign or reflection of a dysfunction in neuroimmune-endocrine regulations.