Thermal Withdrawal Thresholds Research Articles

Model of neuropathic intermittent claudication in the rat: methodology and application

In the present study we characterize a rat neurogenic intermittent claudication model which was accomplished by placing two pieces of silicone rubber of various sizes into the lumbar (L4 and L6) epidural space. After induction of spinal stenosis walking function was measured using a treadmill apparatus and sensory functions were tested by measuring thermal and tactile withdrawal threshold (von Frey filaments) for the period of 28 days after stenosis. In addition, local spinal cord blood flow (SCBF) was measured, periodically, before and after induction of stenosis using laser Doppler. After implantation of two pieces of silicone rubber (width 1.25 mm, height 1.0 mm, length 4.0 mm) a significant running dysfunction, as evidenced by shortening of running distance, was measured as soon as 24 h after stenosis (178.5±59.1 m vs 681.3±70.2 m). This effect persisted for 28 days after surgery. Similarly, a significant tactile (but not thermal) hypersensitivity was measured for a period of 28 days (1.2±0.3 g vs 14.9±0.2 g). In this experimental group the measurement of local SCBF revealed a significant (30–50%) reduction in the territory of spinal stenosis measured at 3, 7, 14 or 28 days after surgery. Implantation of larger pieces of silicon rubber (1.5 mm width) caused a significant increase in the incidence of urinary retention and mortality rate. These data show that chronic partial spinal compression at L4 and L6 spinal level lead to the development of significant motor/sensory dysfunction which resemble those seen in patients with neurogenic intermittent claudication. The lack of motor dysfunction under resting conditions but its appearance during forced exercise also suggest that the development of local spinal ischemia can represent one of the mechanisms.

EFFECTS OF NEUTRALIZING ANTIBODIES TO TNF‐ALPHA ON PAIN‐RELATED BEHAVIOR AND NERVE REGENERATION IN MICE WITH CHRONIC CONSTRICTION INJURY

Inhibition of proinflammatory cytokines reduces hyperalgesia in animal models of painful neuropathy. We set out to investigate the consequences of this treatment for nerve regeneration. Here we examined the sequels of epineurial application of neutralizing antibodies to tumor necrosis factor‐alpha (TNF) in chronic constriction injury (CCI) of the sciatic nerve in C57/BL 6 mice. The mice were tested behaviorally for manifestations of thermal hyperalgesia and mechanical allodynia. Nerve regeneration was assessed by morphometry of myelinated nerve fibers in the sciatic nerve and of the epidermal innervation density in the glabrous skin of the hindpaws. Antibodies to TNF reduced thermal hyperalgesia and mechanical allodynia after CCI. Myelinated fiber density in the sciatic nerve was reduced to 30% of normal on day 7 after surgery, and reached 60% on day 45, with no difference between antibody‐treated and untreated animals. Epidermal innervation density as shown by PGP 9.5 and CGRP immunohistochemistry was reduced to 25–47% at both time points after CCI, again without differences between antibody treated and untreated mice. Myelinated fiber density but not epidermal innervation density was correlated to thermal and mechanical withdrawal thresholds. We conclude that neutralization of endoneurial TNF attenuates pain related behavior but has no effect on nerve regeneration. Furthermore, the number of epidermal nerve fibers is not relevant to the magnitude of behavioral hyperalgesia in CCI.

Effects of neutralizing antibodies to TNF-alpha on pain-related behavior and nerve regeneration in mice with chronic constriction injury

Inhibition of proinflammatory cytokines reduces hyperalgesia in animal models of painful neuropathy. We set out to investigate the consequences of this treatment for nerve regeneration. Here we examined the sequels of epineurial application of neutralizing antibodies to tumor necrosis factor-alpha (TNF) in chronic constriction injury (CCI) of the sciatic nerve in C57/BL 6 mice. The mice were tested behaviorally for manifestations of thermal hyperalgesia and mechanical allodynia. Nerve regeneration was assessed by morphometry of myelinated nerve fibers in the sciatic nerve and of the epidermal innervation density in the glabrous skin of the hindpaws. Antibodies to TNF reduced thermal hyperalgesia and mechanical allodynia after CCI. Myelinated fiber density in the sciatic nerve was reduced to 30% of normal on day 7 after surgery, and reached 60% on day 45, with no difference between antibody-treated and untreated animals. Epidermal innervation density as shown by PGP 9.5 and CGRP immunohistochemistry was reduced to 25–47% at both time points after CCI, again without differences between antibody treated and untreated mice. Myelinated fiber density but not epidermal innervation density was correlated to thermal and mechanical withdrawal thresholds. We conclude that neutralization of endoneurial TNF attenuates pain related behavior but has no effect on nerve regeneration. Furthermore, the number of epidermal nerve fibers is not relevant to the magnitude of behavioral hyperalgesia in CCI.

Intrathecal treatment with MK-801 suppresses thermal nociceptive responses and prevents c-fos immunoreactivity induced in rat lumbar spinal cord neurons

Sensitization of the second order neurons in the spinal dorsal horn after somatic noxious stimuli is partly mediated by the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor. These neurons also express c-Fos immunoreactivity in response to the somatic noxious stimuli. The present study assessed the inifluence of intrathecal pre-treatment with MK-801, a non-competitive antagonist of NMDA receptor, on thermal sensitization following peripheral noxious heat stimulation. In addition, the influence of MK-801 on c-Fos immunoreactivity in the rat lumbar spinal cord neurons after the peripheral noxious heat was examined. Sprague-Dawley rats were subject to intrathecal catheterization and administration of MK-801 or saline before and after noxious heat (52° C) stimulation of rat hind paws. Thermal sensitization was tested after MK-801 (0.1 μmol 10 μl–1). Fos-like immunoreactivity was evaluated 2 h after noxious stimulation in a separate group of animals. MK-801 significantly increased the thermal withdrawal threshold by 60% following noxious heat stimulation and reduced c-Fos immunoreactivity in the second order neurons by 70% in the dorsal horn. The study suggests that glutamate plays a pivotal role in the thermal nociceptive pathway and indicates that the NMDA receptor is necessary to maintain normal thermal sensitization, possibly by regulating c-fos gene expression in second order neurons. [Neurol Res 1999; 21: 593–598]

Distinct neurochemical features of acute and persistent pain.

To address the neurochemistry of the mechanisms that underlie the development of acute and persistent pain, our laboratory has been studying mice with deletions of gene products that have been implicated in nociceptive processing. We have recently raised mice with a deletion of the preprotachykinin-A gene, which encodes the peptides substance P (SP) and neurokinin A (NKA). These studies have identified a specific behavioral phenotype in which the animals do not detect a window of "pain" intensities; this window cuts across thermal, mechanical, and chemical modalities. The lowered thermal and mechanical withdrawal thresholds that are produced by tissue or nerve injury, however, were still present in the mutant mice. Thus, the behavioral manifestations of threshold changes in nociceptive processing in the setting of injury do not appear to require SP or NKA. To identify relevant neurochemical factors downstream of the primary afferent, we are also studying the dorsal horn second messenger systems that underlie the development of tissue and nerve injury-induced persistent pain states. We have recently implicated the gamma isoform of protein kinase C (PKCgamma) in the development of nerve injury-induced neuropathic pain. Acute pain processing, by contrast, is intact in the PKCgamma-null mice. Taken together, these studies emphasize that there is a distinct neurochemistry of acute and persistent pain. Persistent pain should be considered a disease state of the nervous system, not merely a prolonged acute pain symptom of some other disease conditions.

Effect of intrathecal agmatine on inflammation-induced thermal hyperalgesia in rats

Agmatine, an endogenous ligand, interacts both with the α 2-adrenoceptors and with the imidazoline binding sites. The effect of intrathecally administered agmatine on carrageenan-induced thermal hyperalgesia was investigated by means of a paw-withdrawal test in rats. The effect of agmatine on morphine-induced anti-hyperalgesia was also studied. Intrathecal agmatine in doses larger than 250 μg caused a decrease in the pain threshold, with vocalization and agitation lasting for several hours in all animals. Agmatine alone at 1–100 μg did not give rise to any change in the thermal withdrawal threshold in the contralateral non-inflamed paw. Agmatine pretreatment was found to dose-dependently attenuate the thermal hyperalgesia induced by intraplantar carrageenan. The effect of 100 μg agmatine was completely lost by 60 min, whereas the effect of 50 μg was of similar magnitude but exhibited a longer duration. Agmatine posttreatment had a slighter effect. Agmatine pretreatment (100 μg) together with 1 μg morphine (subeffective dose) has significantly higher anti-hyperalgesic effect then the individual compounds by themselves. These are the first data demonstrating the behavioral and anti-hyperalgesic effects of intrathecal agmatine. The results reveal important interactions between intrathecal agmatine and opioids in thermal hyperalgesia.

Nerve growth factor evokes hyperalgesia in mice lacking the low-affinity neurotrophin receptor p75

Endogenous nerve growth factor (NGF) has been shown to be an important mediator of inflammatory pain and exogenous application of recombinant human NGF (rhNGF) produces pain and hyperalgesia in animals and humans. Since NGF can act through two receptors types, the high affinity tyrosine kinase A (trkA) receptor and the low affinity p75 receptor, we used transgenic mice lacking p75 to analyse the relative importance of these receptors. After systemic injection of rhNGF (5 mg/kg), pharmacokinetic studies revealed similar serum levels and elimination profiles of exogenously administered rhNGF in both strains of mice. Although animals lacking p75 have increased mechanical and thermal withdrawal thresholds they developed both heat and mechanical hyperalgesia after systemic injection of rhNGF whose magnitude did not differ significantly from wildtype animals. This means that NGF-induced hyperalgesia can occur in the absence of the p75 receptor and suggests that the trkA receptor is sufficient to mediate the acute noxious action of NGF.

Force of limb withdrawals elicited by graded noxious heat compared with other behavioral measures of carrageenan-induced hyperalgesia and allodynia

We investigated if carrageenan-induced thermal hyperalgesia and mechanical allodynia are associated with a reduction in threshold and–or enhancement of suprathreshold nocifensive responses, using a method to measure the force of a hind limb withdrawal reflex elicited by graded noxious heat stimuli (36–52°C, 5 s) delivered by Peltier thermode tethered to the ventral hind paw of conscious rats. Withdrawal reflexes were recorded 2.5 h after intraplantar injection of carrageenan (1 or 0.1%) or saline vehicle in sessions >2 weeks apart: baseline reflexes were assessed the day before. Withdrawal reflex force increased linearly from 42–52°C. Carrageenan 1% significantly enhanced withdrawal reflexes at 40–46°C, reducing the slope and threshold of the stimulus-response function. This was associated with significant reductions in thermal paw withdrawal latency (Hargreaves test: by 50%), mechanical withdrawal threshold (by 82%) and weight bearing on the injected side (by 81%) measured with independent force plates. Smaller reductions in thermal paw withdrawal latency and mechanical withdrawal threshold, and smaller enhancement of withdrawal reflex force, were observed following 0.1% carrageenan. Intraplantar saline was ineffective. This method allows assessment of hyperalgesic changes in stimulus-response coding over a broad range of noxious stimulus intensities.

Leukemia inhibitory factor induces mechanical allodynia but not thermal hyperalgesia in the juvenile rat

Systemic administration or local injection to the rat hindpaw of leukemia inhibitory factor induced a prolonged, dose dependent, mechanical hypersensitivity of the hindpaw flexion withdrawal reflex. Mechanical stimuli which were innocuous prior to leukemia inhibitory factor administration, evoked a rapid hindpaw withdrawal reflex indicative of mechanical allodynia. Pre-administration of anti-leukemia inhibitory factor antibodies prevented this behavioural hypersensitivity. Hindpaw sensitivity to a noxious thermal stimulus was unaffected by leukemia inhibitory factor administration. Anti-leukemia inhibitory factor had no effect upon hindpaw withdrawal thresholds when injected alone nor influenced the mechanical hypersensitivity produced by a subcutaneous injection of nerve growth factor. Injection of the closely related cytokine ciliary neurotrophic factor did not affect mechanical or thermal reflex withdrawal thresholds. Elevation of the neuroactive cytokine leukemia inhibitory factor following peripheral nerve injury may be a contributory factor to the pathogenesis of neuropathic pain.

The role of nitric oxide in the development and maintenance of the hyperalgesia produced by intraplantar injection of carrageenan in the rat

Activation of the N-methyl- d-aspartate (NMDA) receptor has been reported to be involved in the mechanisms that underlie thermal hyperalgesia produced by the intraplantar injection of carrageenan. As NMDA-mediated thermal hyperalgesia produced in models of acute and persistent pain have been reported to involve production of nitric oxide, we examined the role of nitric oxide in both the development and maintenance of the thermal hyperalgesia produced by the intraplantar injection of carrageenan. In addition, we examined the role of nitric oxide in the maintenance of the mechanical hyperalgesia produced by intraplantar injection of carrageenan. Prior to the intraplantar injection of carrageenan (2mg in 100 μ1) there was no significant difference in thermal withdrawal latencies or mechanical withdrawal thresholds between the left and right hindpaws. Three hours after injection of carrageenan into the left hindpaw, rats showed evidence of a significantly faster thermal withdrawal latency and lower mechanical withdrawal threshold of the left hindpaw compared to the right hindpaw. In addition, the left hindpaw was significantly increased in size (diameter) compared with the right hindpaw. In these same rats, the intrathecal administration of saline, N G-nitro-l-arginine methyl ester ( l-NAME; 2–200 nmol) or the inactive enantiomer, N G-nitro-d-arginine methyl ester ( d-NAME; 200 nmol) did not produce any significant change in thermal nociceptive withdrawal latencies in the non-injected paw. However, administration of l-NAME (2–200 nmol), but not saline or d-NAME, produced a dose dependent and reversible block of the thermal hyperalgesia for a period of up to 3 h. In contrast, neither l-NAME, d-NAME nor saline affected the mechanical hyperalgesia or the edema produced by the intraplantar injection of carrageenan. These data suggest that the production of nitric oxide, in the spinal cord, is involved in the maintenance of the thermal hyperalgesia, but not the mechanical hyperalgesia or the inflammation produced in this model of persistent pain. As these experiments demonstrated that nitric oxide was involved in the maintenance of thermal but not mechanical hyperalgesia, an additional series of experiments was conducted to examine the role of nitric oxide in the development of the thermal hyperalgesia produced by the intraplantar injection of carrageenan. Administration of l-NAME (2–200 nmol), but not d-NAME (200 nmol) or saline, 30 min prior to carrageenan and again at 90 min after carrageenan produced a delay in the onset of the thermal hyperalgesia, consistent with the time course of the effect of l-NAME. That is, blocking the production of nitric oxide resulted in a delay in the onset of thermal hyperalgesia, but did not prevent its development. Collectively, these data are consistent with the hypotheses that the maintenance of thermal but not mechanical hyperalgesia is dependent on production of nitric oxide, and that the development of thermal hyperalgesia involves the production of nitric oxide.