Immersion Of Paw Research Articles

Neurofilament light chain in plasma as a sensitive diagnostic biomarker of peripheral neurotoxicity: In Vivo mouse studies with oxaliplatin and paclitaxel - NeuroDeRisk project

Identifying compounds that are neurotoxic either toward the central or the peripheral nervous systems (CNS or PNS) would greatly benefit early stages of drug development by derisking liabilities and selecting safe compounds. Unfortunately, so far assays mostly rely on histopathology findings often identified after repeated-dose toxicity studies in animals. The European NeuroDeRisk project aimed to provide comprehensive tools to identify compounds likely inducing neurotoxicity. As part of this project, the present work aimed to identify diagnostic non-invasive biomarkers of PNS toxicity in mice. We used two neurotoxic drugs in vivo to correlate functional, histopathological and biological findings. CD1 male mice received repeated injections of oxaliplatin or paclitaxel followed by an assessment of drug exposure in CNS/PNS tissues. Functional signs of PNS toxicity were assessed using electronic von Frey and cold paw immersion tests (oxaliplatin), and functional observational battery, rotarod and cold plate tests (paclitaxel). Plasma concentrations of neurofilament light chain (NF-L) and vascular endothelial growth factor A (VEGF-A) were measured, and histopathological evaluations were performed on a comprehensive list of CNS and PNS tissues. Functional PNS toxicity was observed only in oxaliplatin-treated mice. Histopathological findings were observed dose-dependently only in paclitaxel groups. While no changes of VEGF-A concentrations was recorded, NF-L concentrations were increased only in paclitaxel-treated animals as early as 7 days after the onset of drug administration. These results show that plasma NF-L changes correlated with microscopic changes in the PNS, thus strongly suggesting that NF-L could be a sensitive and specific biomarker of PNS toxicity in mice.

Topical treatment with a transient receptor potential ankyrin 1 (TRPA1) antagonist reduced nociception and inflammation in a thermal lesion model in rats

Thermal injury promotes tissue inflammation and pain, which is difficult to control. Different peripheral mechanisms seem to be involved in burn pain, such as free radical-induced damage, but further study is still needed to understand how oxidant substances induced nociceptor sensitization. The transient receptor potential ankyrin 1 (TRPA1) is an ion channel activated by oxidants substances, and it could be sensitized after tissue inflammation. This study evaluated the TRPA1 involvement in nociception and inflammation produced by a thermal injury model. Male Wistar rats were used. The concentration of the TRPA1 antagonist (HC-030031, 0.05%) on base cream was chosen using allyl isothiocyanate intraplantar test. Then, the base cream containing HC-030031 was tested on the thermal injury model (induced by warm water immersion of hind paw, under anesthesia), and silver sulfadiazine (1%) was used as a positive control. Cream treatments on the hind paw were done daily (200 mg/paw) for 6 days after thermal injury. Also, nociception (static and dynamic mechanical allodynia, heat allodynia, and spontaneous pain) or edema were evaluated. On day 6, inflammatory and oxidative parameters were assessed. The base cream containing HC-030031 produced antinociceptive and anti-inflammatory effects (reduced the edema and inflammatory cells infiltration) and decreased the levels of hydrogen peroxide, or superoxide dismutase and NADPH oxidase activities after thermal injury. Thus, this study showed the involvement of the TRPA1 receptor in the nociception and inflammation caused by thermal injury and suggested that TRPA1 antagonists might be useful as novel treatments for pain and inflammation by topical application.

Evaluation of nerve growth factor (NGF) treated mesenchymal stem cells for recovery in neurotmesis model of peripheral nerve injury

BackgroundPeripheral nerve damages are a relatively common type of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, the extent of regeneration is not remarkable. The present study aimed to evaluate the effect of NGF treated mesenchymal stem cells on regeneration of transected sciatic nerve. Materials and methodsIn this experimental study, forty-two male Wistar.rats (180–200 g) were randomly divided into 6 groups (n = 7) including control, Membrane + Cell (Mem + Cell), NGF group, NGF + Cell group, NGF + Mem group and NGF + Mem + Cell group. Regeneration of sciatic nerve was evaluated using behavioral analysis, electrophysiological assessment and histological examination. ResultsThe rats in the NGF + Mem + Cell group showed significant decrease in sciatic functional index (SFI) and hot water paw immersion test during the 2nd to 8th weeks after surgery. (p < 0.001). At 8 weeks after surgery, electrophysiological findings showed that amplitude increased and latency decreased significantly in NGF + Mem + Cell group (p < 0.001). Measured histological parameters showed that number of nerve fibers, number of vessels and percent of vessel area also increased significantly in NGF + Mem + Cell group (p < 0.05). ConclusionThe present study showed that NGF in accompany with mesenchymal stem cells improved electrophysiological and histological indices.

Chitosan-film associated with mesenchymal stem cells enhanced regeneration of peripheral nerves: A rat sciatic nerve model

ObjectivePeripheral nerve injuries comprise significant portion of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, but the extent of regeneration is not remarkable. Regeneration might be through the activity of the mesenchymal stem cells (MSCs) which can release growth factors or extracellular matrix components or by the therapeutic effect of some material with the MSCs. The present study aimed to evaluate the regeneration of transected sciatic nerve by a therapeutic value of mesenchymal stem cells (MSCs) associated with chitosan-film (Cs) in rat. Materials & methodsMale Wistar rats (n=42, 180-200g) were randomly divided into intact; control; sham; Cs; MSCs; MSCs + Cs groups. Functional recovery was evaluated at 2, 4, 6 and 8 weeks after surgery using sciatic functional index (SFI), hot water paw immersion test, electrophysiological, histological analyses. ResultsThe rats in the MSCs+Cs group showed significant decrease in SFI and hot water paw immersion test during the 2nd to 8th weeks after surgery. Electrophysiological findings showed a significant decrease in latency time in the MSCs +Cs group. Amplitude of the nerve impulses also increased. Number of nerve fibers with more than 6 μm diameters increased significantly in MSCs+Cs. The number of nerve fibers with less than 4 μm diameters also increased significantly in MSCs+Cs group. ConclusionTaken together, mesenchymal stem cells associated with Cs could improve functional and histomorphological properties of the sciatic nerve after injury which may have some clinical outcomes as well.

A simple, sensitive and accurate method for rat paw volume measurement and its expediency in preclinical animal studies

Several methods are used to evaluate the inflammatory changes. Measurement of edema is the most commonly used method for evaluating artificially induced inflammation in rat model. In this context, we present a method for the measurement of volume of rat paw, in which two glass columns containing fluid are connected by a glass tube. Paw is immersed in one column and the other column is placed on weighing balance. Upon immersion of paw, equal volume of fluid displaced applies a force F, which shows specific and proportional increment in the level of fluid in both the columns, which could then be detected by weighing balance, as 'Force = Weight' of displaced fluid, using the specific gravity of the fluid and the volume of paw, and thus the change in the volume of paw can be calculated. The rat paw immersed in column without touching the wall of the column facilitates the accurate measurement of volume of paw when measured several times. Present method is validated for its accuracy and reproducibility when internal radii of both the columns were same or different. Presented method is also cost-effective and hence can be used for the academic and the commercial animal research purpose, without compromising the precision of the evaluation.

PGE 2-induced lasting nociception to heat: Evidences for a selective involvement of A-delta fibres in the hyperpathic component of hyperalgesia

Animal models for mechanical pressure or heat nociception usually only measure the threshold response latency. In this study, the effect of typical sensitising treatments on the lasting nocifensive behaviour elicited after a supra-threshold heating stimulus – the hyperpathic component of hypernociception – was assessed. Male Wistar rats received either intra-plantar (i.pl.) injection of 350 ng PGE 2 (50 μL) or topical application (t.a.) of 100% dimethylsulfoxide (DMSO), and 10 mM capsaicin. One hour after the paw treatments the number of nocifensive events (NNE) was scored hourly (6 h), for 5 min, immediately after a hind paw immersion in hot water (50 °C/7 s). PGE 2, DMSO and capsaicin increased the NNE -induced by the supra-threshold stimuli. Indomethacin (2.5 mg/kg i.p.), given 30 min before paw treatments, completely inhibited NNE in all groups ( P < 0.01). However, indomethacin given 60 min after PGE 2 did not reverse this sensitisation. PGE 2 and DMSO did not lower the heat threshold in the paw withdrawal test, although carrageenan and capsaicin were effective ( P < 0.05). Capsaicin neonatal treatment (CNT) (50 mg/kg) reduced the sensitisation induced by DMSO and capsaicin ( P < 0.01), but not that induced by PGE 2. These data suggest that the heat-induced lasting nociception is probably conveyed by Að nociceptors, and PGE 2 seems to be more selective to induce this phenomenon than the thermal threshold lowering. In addition, this hyperpathic effect induced by DMSO and capsaicin seems to be indirectly mediated by PGE 2 and C-fibres.

Preemptive effects of intrathecal cyclooxygenase inhibitor or nitric oxide synthase inhibitor on thermal hypersensitivity following peripheral nerve injury

The present study provides an important implication for the management of chronic neuropathic pain, focusing on prostaglandin (PG) and nitric oxide (NO) in the spinal cord. To determine if spinally administered cyclooxygenase (COX) inhibitor or nitric oxide synthase (NOS) inhibitor had preemptive analgesia on thermal hypersensitivity induced by chronic constrictive nerve injury, Sprague-Dawley rats were chronically implanted with an intrathecal (i.t.) catheter. The left sciatic nerve was loosely ligated with 2-mm polyethylene tubing to produce painful mononeuropathy. Animals received tenoxicam (7.5, 15 or 30 μmol/10 μl, i.t.), NS-398 (15 or 30 μmol), or L-NAME (30, 150 or 300 μmol) immediately before the nerve injury, followed by daily injection extending into the four postoperative days. The hindpaw was immersed into a hot (42°C, 44°C and 46°C) or cold (10°C) water bath. The paw immersion test revealed significant thermal hyperalgesia and allodynia 5 day after nerve injury in vehicle control animals. Tenoxicam (7.5, 15 or 30 μmol) or L-NAME (30, 150 or 300 μmol) dose-dependently attenuated hyperalgesia and allodynia. Equimolar dose of NS-398 (15 or 30 μmol) also diminished these nociceptive behaviors. Higher dose of either drug primarily produced longer duration of inhibition. The inhibitory effect of tenoxicam (30 μmol) on hyperalgesia was more effective than that of an equimolar dose of NS-398 or L-NAME. These results demonstrated that intrathecally administered COX inhibitor or NOS inhibitor provides preemptive analgesia on thermal hypersensitivity following chronic constrictive nerve injury in rats.

Growth hormone releasing hormone reverses endotoxin-induced localized inflammatory hyperalgesia without reducing the upregulated cytokines, nerve growth factor and gelatinase activity

During inflammatory processes, the hypothalamic-pituitary axis is activated which can subsequently result in analgesia. For example, hypothalamic corticotrophin-releasing hormone (CRH) that is released during such activation has been attributed with analgesic actions. It is believed that the somatotrophic axis is also activated during inflammation. The aim of this study was to determine the analgesic actions of growth hormone-releasing hormone (GHRH), in a rat model of localized inflammatory hyperalgesia, induced by intraplantar (i.pl.) endotoxin (ET) injections. Pretreatment with intraperitoneal (i.p.) injections of GHRH (2, 5, 10 μg kg −1) 30 min before i.pl. ET injection (1.25 μg in 50 μl saline) prevented, in a dose-dependent manner, both mechanical hyperalgesia determined by the paw pressure (PP) test and thermal hyperalgesia determined by the hot plate (HP) and paw immersion (PI) tests. Pretreatment with GHRH had no significant effect on the elevated levels of the inflammatory mediators, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and nerve growth factor (NGF) due to i.pl. ET injection. No significant effect was obtained by pretreatment with GHRH, on the increased expression of gelatinase B due to ET injection. In conclusion, GHRH reverses inflammatory hyperalgesia in the rat without affecting the upregulated inflammatory mediators and these actions may be clinically important.

Responses of C-fiber low threshold mechanoreceptors and nociceptors to cold were facilitated in rats persistently inflamed and hypersensitive to cold

Cold allodynia is an annoying symptom in conditions of chronic inflammation such as rheumatoid arthritis. To examine whether primary afferent nerve activities are changed in association with hypersensitivity to cold, we recorded single nerve activities from the sural nerve in persistently inflamed rats in vivo. Inflammation was induced by an injection of complete Freund’s adjuvant (CFA) solution into the tibio-tarsal joint. Inflamed rats showed an increased number of paw shakes to paw immersion in 25 °C water (pre-inflammation: 1.15±0.58, 2-week inflammation: 4.70±1.15). We also recorded cutaneous C-fiber activities under pentobarbital anesthesia and studied their responses to thermal and mechanical stimuli. The response of C-low threshold mechanoreceptors to cooling (total discharges between 27 and 23 °C) increased 1.8-fold (control group: 5.17±1.04 impulses, inflamed group: 9.38±1.47 impulses). In addition, the proportion of C-nociceptor units responding to cold down to 2 °C was significantly greater in the inflamed group (9 out of 18 units; threshold: 10.0±2.6 °C) than in the intact group (1 out of 14 units; threshold: 4.0 °C). These results suggest that the facilitated responses of these primary afferents are associated with cold hypersensitivity in chronically inflamed conditions.

Effects of the novel analgesic, cizolirtine, in a rat model of neuropathic pain

Cizolirtine (5-{[( N, N-dimethylaminoethoxy)phenyl]methyl}-1-methyl-1 H-pyrazol citrate) is a centrally acting analgesic with a currently unknown mechanism of action, whose efficacy has been demonstrated in various models of acute and inflammatory pain in rodents. Further studies were performed in order to assess its potential antinociceptive action in a well-validated model of neuropathic pain, i.e. that produced by unilateral sciatic nerve constriction in rats. Animals were subjected to relevant behavioural tests based on mechanical (vocalization threshold to paw pressure) and thermal (struggle latency to paw immersion in a cold (10°C) water bath) stimuli, 2 weeks after sciatic nerve constriction, when pain-related behaviour was fully developed. Acute pretreatment with 2.5–10 mg/kg p.o. of cizolirtine reversed both mechanical and thermal allodynia. These effects were antagonized by prior injection of the α 2-adrenoceptor antagonist idazoxan (0.5 mg/kg i.v.), but not the opioid receptor antagonist naloxone (0.1 mg/kg i.v.). On the other hand, cizolirtine (10 mg/kg p.o.) produced no motor deficits in animals using the rotarod test. Our study showed that cizolirtine suppressed pain-related behavioural responses to mechanical and cold stimuli in neuropathic rats, probably via an α 2-adrenoceptor-dependent mechanism. These results suggest that cizolirtine may be useful for alleviating some neuropathic somatosensory disorders, in particular cold allodynia, with a reduced risk of undesirable side effects.

Antinociceptive effect of systemic gabapentin in mononeuropathic rats, depends on stimulus characteristics and level of test integration

The anticonvulsant gabapentin is effective against neuropathic pain, but the primary site(s) and mechanism(s) of action are unknown. In order to explore the relative contribution of spinal versus supra-spinal mechanisms to the antinociceptive effect of gabapentin, this study used two differentially integrated nociceptive tests. We systematically compared the effects of various doses of gabapentin on the paw withdrawal to pressure (PWTP), a spinally coordinated reflex and the vocalization threshold to paw pressure (VTPP), a supra-spinal integrated test in the sciatic nerve constriction rat model of neuropathic pain. In addition, we evaluated the effect of gabapentin on the struggle latency to paw immersion into a non-noxious cold (10°C) water bath. Similar lower doses (1–30 mg/kg) of gabapentin produced potent antinociception in the VTPP test but were devoid of effects on the PWTP. The effect was observed not only on the nerve-injured side, but also, although less pronounced, on the contralateral side. Only the highest dose (100 mg/kg) of the anticonvulsant was able to induce an increase in the nerve-injured paw threshold in both tests. In the thermal test, gabapentin (3, 10 and 30 mg/kg i.p.) dose-dependently increased the response time to the 10°C stimulus. Gabapentin at 100 mg/kg but not at 30 mg/kg produced motor deficits in animals using the rotarod test. Taken together, our findings suggest that low doses of gabapentin have a preferential action on the more integrated pain-related behaviour in neuropathic rats. The present results confirm that gabapentin may be a useful approach for the clinical management of several aspects of neuropathic pain.

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-1β in the liver. In this study, we demonstrate that such injections of thymulin result in a significant elevation in the levels of TNF-α ( 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-1β. Pretreatment with specific antagonists to each of these factors (polyclonal anti-TNF-α, 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.

Complete prevention but stimulus-dependent reversion of morphine tolerance by the glycine/NMDA receptor antagonist (+)-HA966 in neuropathic rats.

Tolerance to the analgesic effect of morphine complicates the management of chronic pain states. The authors studied the ability of the glycine/N-methyl-D-aspartate receptor antagonist (+)-HA966 to modify morphine tolerance in a rat model of neuropathic pain. Mononeuropathy was induced by placing four ligatures around the common sciatic nerve. The 4-day pretreatment regimens with two daily subcutaneous injections of saline and saline, saline and morphine (10 mg/kg), (+)-HA966 (2.5 mg/kg) and morphine, or (+)-HA966 and saline were begun on post-operative day 12 to test the ability of (+)-HA966 to prevent the development of tolerance. Behavioral experiments were performed on postoperative day 16, when the pain-related behavior reached a stable maximum. The effect of an acute dose of morphine (1 mg/kg intravenously) was tested against both mechanical (vocalization threshold to paw pressure) and thermal (struggle latency to hind paw immersion into a 46 degrees C hot-water bath) stimuli. In addition, to test the ability of a single injection of (+)-HA966 to reverse established morphine tolerance, groups of morphine-pretreated rats received injections of either (+)-HA966 (2.5 mg/kg subcutaneously) and morphine (1 mg/kg intravenously), saline and morphine, or (+)-HA966 and saline. Baseline vocalization thresholds and struggle latencies did not differ in the various pretreatment groups, indicating that the pretreatments had no effect on pain-related behavior. Coadministration of (+)-HA966 prevented the reduction of the effect observed with morphine alone in both the mechanical test and the thermal test. (+)-HA966 reversed morphine tolerance in the thermal but not in the mechanical test. (+)-HA966 prevented morphine tolerance in both mechanical and thermal tests but reversed established morphine tolerance in the thermal test only.

Pain-Induced Analgesia Mediated by Mesolimbic Reward Circuits

We tested the hypothesis that noxious stimuli induce pain modulation by activation of supraspinal structures. We found that intense noxious stimuli (i.e., subdermal injection of capsaicin or paw immersion in hot water) induced profound attenuation of the jaw-opening reflex in the anesthetized rat; forepaw subdermal capsaicin also elevated the mechanical hindpaw-withdrawal threshold in the awake rat. These antinociceptive effects were blocked by previous injection of either a dopamine antagonist (flupentixol) or an opioid antagonist (naloxone) into the nucleus accumbens. Additional experiments in anesthetized animals showed that the antinociceptive effect of noxious stimulation by either capsaicin (>/=100 micrograms) or hindpaw immersion in hot water (>/=45 degrees C for 4 min) correlated with the intensity of the stimulus. The maximal antinociceptive effect of capsaicin was similar in magnitude to that of a high dose of morphine (10 mg/kg) injected subcutaneously. Injection of the GABA(A)-receptor agonist muscimol, but not naloxone, into the rostroventral medulla, a major component of descending pain modulation systems, blocked capsaicin-induced antinociception. Although it is widely thought that painful stimuli may induce analgesia by activating forebrain structures, this is the first demonstration that such a mechanism exists. Furthermore, this mechanism can be engaged by naturalistic stimuli in awake animals. These observations imply that painful stimuli might under certain conditions be rewarding.

Involvement of Interleukin-1β, Nerve Growth Factor, and Prostaglandin-E2in 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-1β (IL-1β) 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-1β and PGE2induced 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-1β mechanisms.

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.

Relationship between mechano-receptive fields of dorsal horn convergent neurons and the response to noxious immersion of the ipsilateral hindpaw in rats

This study examines the relationship between mechano-receptive fields (inhibitory and excitatory, located on the ipsilateral hindpaw) of convergent dorsal horn neurons, and the responses of the neurons to noxious immersion of an entire paw in noxious hot water. In pentobarbital anesthetized rats with intact spinal cords and in unanesthetized decerebrate-spinalized rats, rat hindpaws were immersed in 50°C water for 10 s after the mechano-receptive fields had been delineated using 5-s noxious pinches. Convergent neurons were either excited or inhibited by noxious immersion of the hindpaw. In both groups, a significant association ( χ 2, P<0.01) was found between the make-up of the mechano-receptive field and the response of the neuron to immersion. Immersion-inhibited neurons (intact=27, spinalized=13), always had both an excitatory and an inhibitory mechano-receptive field on the same hindpaw. Additionally, when the hindpaw was removed from the noxious water, these immersion-inhibited cells displayed a strong afterdischarge which was immediately inhibited once the paw was reimmersed. Pinch-induced and immersion-induced inhibition were found in both spinalized and intact rats suggesting spinal mechanisms were sufficient to mediate this effect. The majority of immersion-excited cells showed only an excitatory mechano-receptive field on the hindpaw (intact rats=18/23 or 78.3%, spinalized rats=24/36 or 66.7%). However, other immersion-excited cells had both an inhibitory and an excitatory mechano-receptive field on the hindpaw (intact rats=5/23 or 21.7%, spinalized rats=12/36 or 33.3%). The response of a convergent neuron, which has its excitatory receptive field located on a paw, to noxious immersion of the entire paw can be predicted by the make-up of the mechano-receptive fields. Additionally, since noxious paw immersion affects ipsilateral convergent neurons in two opposite manners, it suggests that other effects, such as heterotopic actions, might also not be uniform.

Augmentation of nociceptive reflexes and chronic deafferentation pain by chemical lesions of either dopaminergic terminals or midbrain dopaminergic neurons

Neurodegenerative diseases affecting the midbrain dopaminergic system have been reported to produce spontaneous pains like in Parkinson's disease. Using various pain tests for acute (hot plate test, HPT, tail flick, TFT, paw pressure test, PPT and paw immersion test, PIT) and chronic deafferentation (autotomy, AT, following peripheral neurectomy) pains in rats, we have investigated the effects on these tests of selective chemical lesions with 6-hydroxydopamine (6-OHDA) or/and kainic acid (KA) either in the striatum or in the substantia nigra (SN) and ventral tegmental area (VTA). 6-OHDA lesions of dopaminergic terminals in the striatum decreased significantly the latencies of all nociceptive reflexes (HPT from 11.7±1.45 s to 7±1.35 s, TFT from 4.5±0.15 s to 3.2±0.16 s and PPT on the contralateral leg from 2.07±0.45 s to 1.05±0.085 s) and accelerated the time of onset (from 10.82±2.3 days to 3.1±0.52 days) and end (from 29.5±5.6 days to 5.2±1.1 days) of AT. These effects were not modified by simultaneous injection of KA and 6-OHDA in the striatum. 6-OHDA lesions in the SN-VTA produced comparable effects to those of similar injections in the striatum, while KA lesions in the SN-VTA did not produce significant changes in the latencies of nociceptive reflexes or in the AT criteria. These results suggest that the dopaminergic system plays a major role in the processing of nociceptive information in the striatum and the limbic areas.

Noxious peripheral stimulation produces antinociception mediated via substance P and opioid mechanisms in the rat tail-flick test

Physiological experiments were run to examine the effects of noxious thermal stimulation of one hindpaw on the tail-flick reflex in the lightly anesthetized rat. Male Sprague-Dawley rats were anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, either a non-noxious or a noxious stimulus was applied which consisted of immersion of one hindpaw in water at 40, 45, 50 or 55°C for 1.5 min. After immersion, tail-flick readings were taken at 3-min intervals for at least 16 min. Paw immersion in water at 55°C induced an antinociceptive response, consisting of an increase in the reaction time, at 0.5 min after immersion. Recovery to baseline levels occurred over the next 3–6 min. Immersion at lower temperatures provoked smaller antinociceptive responses, except at 40°C, where readings remained around the baseline values. The increase in reaction time in response to immersion at 55°C was attenuated or blocked by the novel, nonpeptide substance P (NK-1) receptor antagonist, CP-96,345, administered s.c. 30 or 60 min, respectively, prior to paw immersion. Similar injection of CP-96,344, the inactive stereoisomer, had no effect on the response, while another NK-1 receptor antagonist, CP-99,994, also attenuated the antinociceptive effect of the immersion. The increase in reaction time induced by immersion at 55°C was absent in animals treated neonatally with capsaicin. This nociceptive response was blocked by the broad spectrum opiate antagonist, naloxone (10 mg/kg i.p.) given 20 min before paw immersion. The data provide evidence that a noxious stimulus produces an intensity-related heterosegmental antinociceptive effect and that C-fibers, and NK-1 and opiate receptors are involved in mediating this effect.

Systemic clonidine differentially modulates the abnormal reactions to mechanical and thermal stimuli in rats with peripheral mononeuropathy

The antinociceptive action of the systemically administered α 2-adrenoceptor agonist clonidine was evaluated in a rat model of peripheral unilateral mononeuropathy produced by loose ligatures around the common sciatic nerve, using nociceptive tests based on mechanical (vocalization threshold to paw pressure) or thermal (struggle latency to paw immersion in a cold (10°C) or hot (44°C) water bath) stimuli. Experiments were performed 2–3 weeks after surgery when pain-related behavior was fully developed. We demonstrated a dissociative action depending on the test used: clonidine (3–100 μg/kg i.v.) had a moderate effect on the abnormal reactions to the mechanical stimulus. By contrast it dramatically increased the struggle latency to hot or cold stimuli. These latter effects were completely prevented by prior administration of the α 2-adrenoceptor antagonist idazoxan (0.5 mg/kg i.v.).