Nerve Injury Induces a Tonic Bilateral μ-Opioid Receptor-mediated Inhibitory Effect on Mechanical Allodynia in Mice

Background: Mice lacking the [mu]-opioid receptor gene have been used to characterize the role of [mu]-opioid receptors in nociception and the analgesic actions of opioid agonists. In this study, the authors determined the role of [mu]-opioid receptors in neuropathic pain behaviors and the effectiveness of [mu]- and [kappa]-opioid receptor agonists on this behavior in mice.

Methods: The authors studied the behavioral responses of [mu]-opioid receptor knockout and wild-type mice to thermal and mechanical stimuli before and after neuropathic pain induced by unilateral ligation and section of the L5 spinal nerve. Response to mechanical stimuli was evaluated by determining the frequency of hind paw withdrawal to repetitive stimulation using a series of von Frey monofilaments. Thermal hyperalgesia was assessed by determining the paw withdrawal latencies to radiant heat and frequency of hind paw withdrawal to cooling stimuli. The effects of systemic morphine, the [kappa]-opioid agonist U50488H, and naloxone on responses to mechanical and thermal stimuli were also studied in spinal nerve-injured mice.

Results: After spinal nerve injury, wild-type mice developed increased responsiveness to mechanical, heat, and cooling stimuli ipsilateral to nerve injury. [mu]-Opioid receptor knockout mice not only had more prominent mechanical allodynia in the nerve-injured paw, but also expressed contralateral allodynia to mechanical stimuli. Hyperalgesia to thermal stimuli was similar between [mu]-opioid knockout and wild-type animals. Morphine decreased mechanical allodynia dose dependently (3-30 mg/kg subcutaneous) in wild-type mice-an effect that was attenuated in the heterozygous mice and absent in the homozygous [mu]-opioid knockout mice. The [kappa]-opioid agonist U50488H (3-10 mg/kg subcutaneous) attenuated mechanical allodynia in wild-type, heterozygous, and homozygous [mu]-opioid mice. Naloxone in wild-type mice resulted in enhanced ipsilateral and contralateral allodynia to mechanical stimuli that resembled the pain behavior observed in [mu]-opioid receptor knockout mice.     

Morphine, oxycodone, methadone and its enantiomers in different models of nociception in the rat

We studied the effects of the commonly used mu-opioid receptor agonists morphine, oxycodone, methadone and the enantiomers of methadone in thermal and mechanical models of acute pain and in the spinal nerve ligation model of neuropathic pain in rats. Subcutaneous administration of morphine, oxycodone, and methadone produced a dose-dependent antinociceptive effect in the tail flick, hotplate, and paw pressure tests. l-methadone, racemic methadone, and oxycodone had a similar dose-dependent antinociceptive effect, whereas the dose-response curve of morphine was shallower. In the spinal nerve ligation model of neuropathic pain, subcutaneous administration of morphine, oxycodone, methadone and l-methadone had antiallodynic effects in tests of mechanical and cold allodynia. l-methadone showed the strongest antiallodynic effect of the tested drugs. d-methadone was inactive in all tests. Morphine 5.0 mg/kg, oxycodone 2.5 mg/kg, and l-methadone 1.25 mg/kg decreased spontaneous locomotion 30 min after drug administration. In conclusion, in acute nociception all mu-opioid receptor agonists produced antinociception, with morphine showing the weakest effect. In nerve injury pain, l-methadone showed the greatest antiallodynic potency in both mechanical and cold allodynia compared with the other opioids. Opioids seem to have different profiles in different pain models. l-methadone should be studied for neuropathic pain in humans.     

Spinal GABA(A) and GABA(B) receptor pharmacology in a rat model of neuropathic pain

BACKGROUND: This study tests the hypothesis that loss of spinal activity of gamma-aminobutyric acid (GABA) contributes to the allodynia and hyperalgesia observed after peripheral nerve injury. METHODS: Intrathecal catheters were implanted in male Sprague-Dawley rats. Antinociception was assessed by measuring withdrawal latency to immersion of the tail in a 52 degrees C water bath. Nerve injury was produced by ligation of the L5 and L6 spinal nerves. Testing was performed 4-14 days after spinal nerve ligation, when tactile allodynia and thermal hyperalgesia were established. Tactile allodynia was quantitated using the threshold to withdrawal of the hind paw on probing with von Frey filaments. Thermal hyperalgesia was quantitated using the latency to withdrawal of the hind paw from radiant heat. Motor function was tested using a rotarod apparatus. RESULTS: Spinal administration of the GABAA receptor antagonist bicuculline or the GABAB receptor antagonist phaclofen produced tactile allodynia and thermal hyperalgesia in normal rats. The GABAB receptor agonist baclofen, administered spinally, produced antinociception in the tail-flick test, whereas the GABAA receptor agonist isoguvacine did not. Isoguvacine and baclofen each reversed tactile allodynia and thermal hyperalgesia produced by spinal nerve ligation. Baclofen but not isoguvacine prolonged thermal withdrawal latency in nerve-injured rats beyond preoperative values. Baclofen but not isoguvacine impaired motor function. CONCLUSIONS: Pharmacologic inhibition of intrinsic GABA tone in normal rats resulted in tactile allodynia and thermal hyperalgesia, consistent with the hypothesis being tested. Exogenous administration of GABA agonists reversed spinal nerve ligation-induced allodynia and hyperalgesia, also consistent with this hypothesis. Isoguvacine produced specific antihyperalgesic and antiallodynic effects, whereas assessment of the effects of baclofen was complicated by motor dysfunction. Spinal GABAA agonists may provide a specific therapy for neuropathic pain.     

Plasticity in action of intrathecal clonidine to mechanical but not thermal nociception after peripheral nerve injury

BACKGROUND: Intrathecal clonidine reduces tactile allodynia in animal models of neuropathic pain, and this effect is blocked by atropine. However, the role of tonic spinal cholinergic activity and its interaction with alpha2-adrenergic systems in normal and neuropathic conditions and to different sensory methods has not been systematically examined. The authors examined cholinergic receptor involvement in thermal and mechanical sensitivity in normal and neuropathic animals and its interaction with intrathecal clonidine. METHODS: Normal rats and rats that received L5/L6 spinal nerve ligation were tested with acute radiant heat, paw pressure, and punctate mechanical stimulation before and after the intrathecal administration of saline, the muscarinic receptor antagonist, atropine, or a toxin to destroy cholinergic neurons, and then after intrathecal clonidine. RESULTS: Atropine, the cholinergic neuronal toxin, and saline did not alter baseline withdrawal thresholds. In nerve-injured rats, neither saline nor atropine altered antinociception from clonidine to a thermal stimulus, but atropine reduced the effect of clonidine to von Frey filament withdrawal threshold (34 +/- 5.6 vs. 14 +/- 5.8 g [mean +/- SEM], saline vs. atropine; P < 0.05) and to withdrawal threshold to paw pressure after clonidine (174 +/- 18 g vs. 137 +/- 16 g, saline vs. atropine; P < 0.05). CONCLUSIONS: These data suggest that after nerve injury, mechanical but not thermal antinociception from intrathecal clonidine relies on a muscarinic interaction, because only mechanical antinociception was antagonized by atropine. These results do not favor a regulation of nociceptive transmission by a tonic release of acetylcholine in nerve-injured rats.     

Plasticity in Action of Intrathecal Clonidine to Mechanical but Not Thermal Nociception after Peripheral Nerve Injury

Background: Intrathecal clonidine reduces tactile allodynia in animal models of neuropathic pain, and this effect is blocked by atropine. However, the role of tonic spinal cholinergic activity and its interaction with [alpha]2-adrenergic systems in normal and neuropathic conditions and to different sensory methods has not been systematically examined. The authors examined cholinergic receptor involvement in thermal and mechanical sensitivity in normal and neuropathic animals and its interaction with intrathecal clonidine.

Methods: Normal rats and rats that received L5/L6 spinal nerve ligation were tested with acute radiant heat, paw pressure, and punctate mechanical stimulation before and after the intrathecal administration of saline, the muscarinic receptor antagonist, atropine, or a toxin to destroy cholinergic neurons, and then after intrathecal clonidine.

Results: Atropine, the cholinergic neuronal toxin, and saline did not alter baseline withdrawal thresholds. In nerve-injured rats, neither saline nor atropine altered antinociception from clonidine to a thermal stimulus, but atropine reduced the effect of clonidine to von Frey filament withdrawal threshold (34 +/- 5.6 vs. 14 +/- 5.8 g [mean +/- SEM], saline vs. atropine;P < 0.05) and to withdrawal threshold to paw pressure after clonidine (174 +/- 18 g vs. 137 +/- 16 g, saline vs. atropine;P < 0.05).     

Antiallodynic effects of systemic and intrathecal morphine in the spared nerve injury model of neuropathic pain in rats

BACKGROUND: The efficacy of opioids for neuropathic pain remains controversial. The effects of morphine on pain behavior were investigated in two animal models of neuropathic pain: the spared nerve injury (SNI) model and the spinal nerve ligation (SNL) model. METHODS: Nerve injuries were created in rats either by tight ligation and section of the left tibial and common peroneal nerves (SNI) or by unilateral ligation of L5 and L6 spinal nerves (SNL). Paw withdrawal threshold to mechanical stimuli was measured using the up-down method in the hairy and glabrous skin territories of the sural nerve for SNI rats or in the mid-plantar paw of SNL rats. RESULTS: Before SNI, the median paw withdrawal thresholds in hairy and glabrous skin were similar (26 g [25%, 75% quartiles: 26, 26 g]). The paw withdrawal threshold decreased after SNI in both hairy and glabrous skin (P < 0.001). Thirty days after the SNI, the threshold in hairy skin (0.3 g) was significantly lower than in glabrous skin (1.9 g; P < 0.001). In blinded experiments, both subcutaneous and intrathecal morphine (0.1-10 microg) dose-dependently attenuated mechanical allodynia induced by SNI measured in the hairy skin, an effect that was naloxone reversible. The ED50 for the intrathecal morphine was 0.52 microg (95% confidence interval, 0.31-0.90 microg). Morphine (1 microg intrathecal) attenuated SNI-induced mechanical allodynia in glabrous skin with potency similar to that in hairy skin. In SNL rats, morphine (30 microg intrathecal) almost completely reversed the SNL-induced mechanical allodynia. CONCLUSIONS: (1) SNI-induced mechanical allodynia is characterized by a lower paw withdrawal threshold in hairy versus glabrous skin; (2) systemic and intrathecal morphine reverse SNI-induced mechanical allodynia in a dose-dependent fashion; and (3) intrathecal morphine also reverses SNL-induced mechanical allodynia. These results suggest that intrathecal opioids are likely to be effective in the treatment of neuropathic pain.     

Intrathecal clonidine reduces hypersensitivity after nerve injury by a mechanism involving spinal m4 muscarinic receptors

alpha2-Adrenergic agonists reduce mechanical and thermal hypersensitivity in animals with nerve injury and effectively treat neuropathic pain in humans. Previous studies indicate a reliance of alpha2-adrenergic agonists in this setting on spinal cholinergic activation and stimulation of muscarinic receptors. The subtype(s) of muscarinic receptors in the spinal cord that produces antinociception in normal animals is controversial, and those involved in reducing hypersensitivity and interacting with alpha2-adrenergic systems after nerve injury are unstudied. To examine this, the left L5 and L6 spinal nerves were tightly ligated in rats, resulting in reduction in withdrawal threshold to punctate mechanical stimuli. Intrathecal clonidine, 15 micro g, returned the withdrawal threshold to normal. Using highly specific m1 and m4 antagonists, we observed no reduction in the effect of clonidine by the m1 antagonist, but inhibition of clonidine's effect by the m4 antagonist. Western analysis revealed no difference in quantitative expression of m1 and m4 receptor protein in the dorsal spinal cord of spinal nerve-injured animals compared with sham-operated controls, suggesting this interaction with m4 receptors does not reflect an increase in receptor expression. IMPLICATIONS: Neuraxial clonidine is an effective adjunct in the treatment of neuropathic pain and increases acetylcholine concentrations in cerebrospinal fluid in humans. These data in animals suggest that spinal m4 type muscarinic receptors are important to the effect of clonidine in treating hypersensitivity to touch after nerve injury.     

Neurokinin-1 receptors are involved in behavioral responses to high-intensity heat stimuli and capsaicin-induced hyperalgesia in mice.

BACKGROUND: The neurokinin-1 (NK-1) receptor and its ligand, substance P, are thought to play important roles in nociception and hyperalgesia. This study evaluated the role of the NK-1 receptor in processing noxious stimuli in normal and inflammatory states. METHODS: Behavioral responses to heat and mechanical and chemical stimuli were studied in NK-1 receptor knockout mice and wild-type control mice. Thermal nociception was evaluated by measuring paw lick or jump latencies to hot plate (52, 55, and 58 degrees C) and paw withdrawal latencies to radiant heat applied to the hind paws. Mechanical nociception was measured by von Frey monofilament applications to the hind paws. Intraplantar capsaicin-induced (10 microg/20 microl) paw licking and mechanical and heat hyperalgesia were compared in NK-1 knockout and wild-type mice. RESULTS: Withdrawal responses to radiant heat (4.3+/-0.18 s for knockout and 4.4+/-0.8 s for wild-type mice) and von Frey monofilaments were similar in knockout and wild-type mice. In the hot plate test, increasing the hot plate temperature from 52 degrees C to 58 degrees C resulted in a decrease in the response latency in the wild-type mice (30.4+/-17.5 s to 15.2+/-6.8 s, P < 0.05), whereas in the knockout mice the response latencies remained constant (28.2+/-19.8 s to 29+/-15.1 s, not significant). Capsaicin-induced paw licking (14.5+/-12.8 s for knockout and 41.3+/-37.3 s for wild-type mice, P < 0.05) and mechanical and heat hyperalgesia were attenuated in the knockout mice. CONCLUSION: NK-1 receptors contribute to the withdrawal responses to high-intensity heat stimuli and to capsaicin-induced mechanical and heat hyperalgesia.     

Spinal GABAA and GABAB Receptor Pharmacology in a Rat Model of Neuropathic Pain

Background: This study tests the hypothesis that loss of spinal activity of [gamma]-aminobutyric acid (GABA) contributes to the allodynia and hyperalgesia observed after peripheral nerve injury.

Methods: Intrathecal catheters were implanted in male Sprague-Dawley rats. Antinociception was assessed by measuring withdrawal latency to immersion of the tail in a 52[degrees]C water bath. Nerve injury was produced by ligation of the L5 and L6 spinal nerves. Testing was performed 4-14 days after spinal nerve ligation, when tactile allodynia and thermal hyperalgesia were established. Tactile allodynia was quantitated using the threshold to withdrawal of the hind paw on probing with von Frey filaments. Thermal hyperalgesia was quantitated using the latency to withdrawal of the hind paw from radiant heat. Motor function was tested using a rotarod apparatus.

Results: Spinal administration of the GABAA receptor antagonist bicuculline or the GABAB receptor antagonist phaclofen produced tactile allodynia and thermal hyperalgesia in normal rats. The GABAB receptor agonist baclofen, administered spinally, produced antinociception in the tail-flick test, whereas the GABAA receptor agonist isoguvacine did not. Isoguvacine and baclofen each reversed tactile allodynia and thermal hyperalgesia produced by spinal nerve ligation. Baclofen but not isoguvacine prolonged thermal withdrawal latency in nerve-injured rats beyond preoperative values. Baclofen but not isoguvacine impaired motor function.     

α2A Adrenoceptors Contribute to Feedback Inhibition of Capsaicin-induced Hyperalgesia

Background: Studies on receptor knockout mice have so far shown that of the three [alpha]2-adrenoceptor subtypes, the [alpha]2A adrenoceptor has a major role in mediating the powerful central analgesia induced by synthetic [alpha]2-adrenoceptor agonists. However, because a knockout of the gene for the [alpha]2A adrenoceptor has produced only little if any change in the pain sensitivity of control, nerve-injured, or inflamed animals, it has not been clear whether activation of [alpha]2A-adrenoceptors by endogenous ligands has a significant pain regulatory role.

Methods: The authors assessed spontaneous pain behavior and mechanical hypersensitivity induced by administration of capsaicin in the colon or paw of [alpha]2A-adrenoceptor knockout mice versus their wild-type controls.

Results: Enhanced pain hypersensitivity was observed in [alpha]2A-adrenoceptor knockout mice 20 min or more after administration of capsaicin, but before, hypersensitivity and spontaneous pain were of equal magnitude in [alpha]2A-adrenoceptor knockout and wild-type mice. When wild-type mice were pretreated with an [alpha]2-adrenoceptor antagonist, capsaicin-induced pain hypersensitivity increased to a level equal to that in [alpha]2A-adrenoceptor knockout mice. Capsaicin-induced hypersensitivity was suppressed in wild-type but not [alpha]2A-adrenoceptor knockout mice by a centrally acting [alpha]2-adrenoceptor agonist, whereas a peripherally acting [alpha]2-adrenoceptor agonist was without effect on hypersensitivity, although it attenuated capsaicin-induced spontaneous pain behavior in wild-type mice.     

Alpha(2A) adrenoceptors contribute to feedback inhibition of capsaicin-induced hyperalgesia

BACKGROUND: Studies on receptor knockout mice have so far shown that of the three alpha2-adrenoceptor subtypes, the alpha(2A) adrenoceptor has a major role in mediating the powerful central analgesia induced by synthetic alpha2-adrenoceptor agonists. However, because a knockout of the gene for the alpha(2A) adrenoceptor has produced only little if any change in the pain sensitivity of control, nerve-injured, or inflamed animals, it has not been clear whether activation of alpha(2A)-adrenoceptors by endogenous ligands has a significant pain regulatory role. METHODS: The authors assessed spontaneous pain behavior and mechanical hypersensitivity induced by administration of capsaicin in the colon or paw of alpha(2A)-adrenoceptor knockout mice versus their wild-type controls. RESULTS: Enhanced pain hypersensitivity was observed in alpha(2A)-adrenoceptor knockout mice 20 min or more after administration of capsaicin, but before, hypersensitivity and spontaneous pain were of equal magnitude in alpha(2A)-adrenoceptor knockout and wild-type mice. When wild-type mice were pretreated with an alpha2-adrenoceptor antagonist, capsaicin-induced pain hypersensitivity increased to a level equal to that in alpha(2A)-adrenoceptor knockout mice. Capsaicin-induced hypersensitivity was suppressed in wild-type but not alpha(2A)-adrenoceptor knockout mice by a centrally acting alpha2-adrenoceptor agonist, whereas a peripherally acting alpha2-adrenoceptor agonist was without effect on hypersensitivity, although it attenuated capsaicin-induced spontaneous pain behavior in wild-type mice. CONCLUSIONS: This study shows that central alpha(2A)-adrenoceptors contribute to feedback inhibition of pain hypersensitivity. Also, alpha(2A)-adrenoceptors are critical for not only somatic but also visceral antinociceptive effects induced by synthetic alpha2-adrenoceptor agonists.     

Chronic intrathecal infusion of minocycline prevents the development of spinal-nerve ligation-induced pain in rats

BACKGROUND AND OBJECTIVES: Minocycline is a second-generation tetracycline with multiple biological effects, including inhibition of microglial activation. Recently, microglial activation has been implicated in the development of nerve injury-induced neuropathic pain. In this study, the authors examined the effects of continuous intrathecal minocycline on the development of neuropathic pain and microglial activation induced by L5/6 spinal-nerve ligation in rats. METHODS: Under isoflurane anesthesia, male Sprague-Dawley rats (200-250 g) received right L5/6 spinal-nerve ligation and intrathecal catheters connected to an infusion pump. Intrathecal saline or minocycline (2 and 6 microg/h) was given continuously after surgery for 7 days (n = 8 per group). The rat right hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before surgery and on days 1 to 7 after surgery. Spinal microglial activation was evaluated with OX-42 immunoreactivity on day 7 after surgery. RESULTS: Spinal-nerve ligation induced mechanical allodynia and thermal hyperalgesia on the affected hind paw of saline-treated rats. Intrathecal minocycline (2 and 6 microg/h) prevented the development of mechanical allodynia and thermal hyperalgesia induced by nerve ligation. It also inhibited nerve ligation-induced microglial activation, as evidenced by decreased OX-42 staining. No obvious histopathologic change was noted after intrathecal minocycline (6 microg/h) infusion. CONCLUSIONS: In this study, the authors demonstrate the preventive effect of continuous intrathecal minocycline on the development of nociceptive behaviors induced by L5/6 spinal-nerve ligation in rats. Further studies are required to examine if continuous intrathecal minocycline could be used safely in the clinical setting.     

Antiallodynic Effects of Systemic and Intrathecal Morphine in the Spared Nerve Injury Model of Neuropathic Pain in Rats

Background: The efficacy of opioids for neuropathic pain remains controversial. The effects of morphine on pain behavior were investigated in two animal models of neuropathic pain: the spared nerve injury (SNI) model and the spinal nerve ligation (SNL) model.

Methods: Nerve injuries were created in rats either by tight ligation and section of the left tibial and common peroneal nerves (SNI) or by unilateral ligation of L5 and L6 spinal nerves (SNL). Paw withdrawal threshold to mechanical stimuli was measured using the up-down method in the hairy and glabrous skin territories of the sural nerve for SNI rats or in the mid-plantar paw of SNL rats.

Results: Before SNI, the median paw withdrawal thresholds in hairy and glabrous skin were similar (26 g [25%, 75% quartiles: 26, 26 g]). The paw withdrawal threshold decreased after SNI in both hairy and glabrous skin (P < 0.001). Thirty days after the SNI, the threshold in hairy skin (0.3 g) was significantly lower than in glabrous skin (1.9 g; P < 0.001). In blinded experiments, both subcutaneous and intrathecal morphine (0.1-10 [mu]g) dose-dependently attenuated mechanical allodynia induced by SNI measured in the hairy skin, an effect that was naloxone reversible. The ED50 for the intrathecal morphine was 0.52 [mu]g (95% confidence interval, 0.31-0.90 [mu]g). Morphine (1 [mu]g intrathecal) attenuated SNI-induced mechanical allodynia in glabrous skin with potency similar to that in hairy skin. In SNL rats, morphine (30 [mu]g intrathecal) almost completely reversed the SNL-induced mechanical allodynia.     

Systemic physostigmine shows antiallodynic effects in neuropathic rats.

The aim of this study was to examine the antiallodynic and antinociceptive effects of subcutaneously administered physostigmine (50, 100, 200 micrograms/kg), compared with morphine (2.5, 5, 10 mg/kg) and NaCl after spinal nerve ligation in rats. The following stimuli were used: acetone (cold allodynia), von Frey hairs (mechanical allodynia), and paw flick test (thermal nociception). Motility boxes were used to investigate the effects of the drugs on motor performance. Physostigmine attenuated both mechanical and cold allodynia dose-dependently but had no effect on the paw flick test. The effect was antagonized by atropine (muscarinic receptor antagonist) but not by mecamylamine (nicotinic receptor antagonist) or naloxone (opioid receptor antagonist). Morphine produced dose-dependent antiallodynic and antinociceptive effects. In the antiallodynic doses, morphine caused severe rigidity. Physostigmine 200 micrograms/kg impaired locomotor activity, but no rigidity was observed. Implications: Physostigmine has different effects on allodynia and nociception, which suggests that different cholinergic (muscarinic) mechanisms may be involved in neuropathic and nociceptive pain.     

Moxonidine, a selective imidazoline-alpha2 -adrenergic receptor agonist, produces spinal synergistic antihyperalgesia with morphine in nerve-injured mice

BACKGROUND: Moxonidine, a novel imidazoline-alpha2-adrenergic receptor-selective analgesic, was recently identified as antinociceptive but has yet to be evaluated in neuropathic pain models. alpha2-adrenergic receptor-selective analgesics, and high-efficacy opioids, effectively inhibit neuropathic pain behaviors in rodents. In contrast, morphine potency and efficacy decreases in states of neuropathic pain, both in rodents and in humans, but may be restored or enhanced by coadministration of morphine with alpha2-adrenergic receptor-selective analgesics. The current experiments extend the evaluation of opioid-coadjuvant interactions in neuropathic subjects by testing the respective antihyperalgesic interactions of moxonidine and clonidine with morphine in a test of mechanical hyperalgesia. METHODS: Nerve-injured mice (Chung model) were spinally administered moxonidine, clonidine, morphine, and the combinations moxonidine-morphine and clonidine-morphine. Hyperalgesia was detected by von Frey monofilament stimulation (3.3 mN) to the hind paws (plantar surface). The ED50 values were calculated and the interactions tested by isobolographic analysis. RESULTS: In nerve-injured mice, moxonidine, clonidine, and morphine all dose-dependently inhibited mechanical hyperalgesia. Furthermore, the combinations of moxonidine-morphine and clonidine-morphine resulted in substantial leftward shifts in the dose-response curves compared with those of each agonist administered separately. The calculated ED50 values of the dose-response curves of these combinations were significantly lower than their corresponding theoretical additive ED50 values. These results confirmed that both interactions were synergistic. CONCLUSIONS: Moxonidine and clonidine both synergize with morphine to inhibit paw withdrawal from nociceptive mechanical stimuli in nerve-injured mice.     

Vanilloid receptor 1-positive neurons mediate thermal hyperalgesia and tactile allodynia.

BACKGROUND CONTEXT: The vanilloid receptor 1 (VR1) is expressed by the type II A-delta and C-fiber neurons, functioning as a molecular integrator for nociception. VR1 can be selectively ablated by resiniferatoxin (RTX), an ultra-potent excitotoxic agonist, when injected into sensory ganglia. PURPOSE: To evaluate the role of the VR1-positive neurons in neuropathic pain. STUDY DESIGN: Photochemical injury to rat sciatic nerve (Gazelius model). METHODS: Two groups of rats underwent the photochemical injury and RTX treatment. RTX was injected in the dorsal root ganglia (DRGs) of the L3, L4, L5, and L6 nerve roots, either after or before the nerve injury. The animals were tested for thermal hyperalgesia (noxious heat stimuli) and mechanical allodynia (von Frey filaments). Immunohistochemical analysis of the DRGs was performed after euthanasia. RESULTS: In the tactile allodynic rats, RTX injection in the DRGs improved the average withdrawal threshold from 1.62 g to 5.68 g. Immunohistochemical labeling showed that almost all VR1-positive neurons were eliminated. When RTX was administrated into the ipsilateral DRGs before the nerve injury, this treatment prevented the development of tactile allodynia in 12 out of 14 rats. Immunohistochemical staining revealed that the VR1-positive neurons were eliminated in the rats that did not develop tactile allodynia, whereas they were still present in the allodynic rats. CONCLUSIONS: VR1-positive neurons are essential for the development of mechanical allodynia. In rats already exhibiting neuropathic pain, the VR1-positive neurons mediate the most sensitive part of mechanical allodynia. RTX injection in sensory ganglia may represent a novel treatment for neuropathic pain.     

Neurokinin-1 Receptors Are Involved in Behavioral Responses to High-intensity Heat Stimuli and Capsaicin-induced Hyperalgesia in Mice

Background: The neurokinin-1 (NK-1) receptor and its ligand, substance P, are thought to play important roles in nociception and hyperalgesia. This study evaluated the role of the NK-1 receptor in processing noxious stimuli in normal and inflammatory states.

Methods: Behavioral responses to heat and mechanical and chemical stimuli were studied in NK-1 receptor knockout mice and wild-type control mice. Thermal nociception was evaluated by measuring paw lick or jump latencies to hot plate (52, 55, and 58[degree sign]C) and paw withdrawal latencies to radiant heat applied to the hind paws. Mechanical nociception was measured by von Frey monofilament applications to the hind paws. Intraplantar capsaicin-induced (10 [micro sign]g/20 [micro sign]l) paw licking and mechanical and heat hyperalgesia were compared in NK-1 knockout and wild-type mice.

Results: Withdrawal responses to radiant heat (4.3 +/- 0.18 s for knockout and 4.4 +/- 0.8 s for wild-type mice) and von Frey monofilaments were similar in knockout and wild-type mice. In the hot plate test, increasing the hot plate temperature from 52[degree sign]C to 58[degree sign]C resulted in a decrease in the response latency in the wild-type mice (30.4 +/- 17.5 s to 15.2 +/- 6.8 s, P < 0.05), whereas in the knockout mice the response latencies remained constant (28.2 +/- 19.8 s to 29 +/- 15.1 s, not significant). Capsaicin-induced paw licking (14.5 +/- 12.8 s for knockout and 41.3 +/- 37.3 s for wild-type mice, P < 0.05) and mechanical and heat hyperalgesia were attenuated in the knockout mice.     

Peripheral block of the hyperpolarization-activated cation current (Ih) reduces mechanical allodynia in animal models of postoperative and neuropathic pain

BACKGROUND AND OBJECTIVES: Block of the hyperpolarization-activated inward current (I h) reduces excitability of peripheral axons during stimulation and decreases ectopic discharges in axotomized sensory neurons. Changes in I h expression in DRG neurons have been suggested to partially underlie sensitization after nerve injury and inflammation. We hypothesized that peripheral block of I h on axons would produce an antiallodynic effect in postoperative as well as neuropathic conditions, and we tested perineural administration of ZD 7288, a specific blocker of I h , on pain-associated behavior in animal models of neuropathic and postoperative pain. METHODS: Under halothane anesthesia, partial sciatic nerve injury or hind-paw incision were performed on adult male rats as previously described. Mechanical allodynia was inferred by demonstration of a decrease in paw withdrawal threshold by application of calibrated von Frey filaments. After surgery, animals received either a saline or a ZD 7288 solution either by sciatic perineural injection or by intraplantar injection. RESULTS: Perineural administration of ZD 7288 (100 microM) significantly reduced mechanical allodynia induced by partial sciatic nerve injury and hind-paw incision. Saline and 10 microM of ZD 7288 had no significant effect on mechanical allodynia. Contralateral administration of ZD 7288, 100 microM, did not affect ipsilateral paw withdrawal threshold after nerve injury. Intraplantar injection of ZD 7288 failed to reduce mechanical allodynia after nerve injury. Sedation and motor effects were not observed. CONCLUSIONS: The current study shows that peripheral block of I h produces an antiallodynic effect, which suggests that I h channels represent a novel target for nerve block treatment of postoperative and neuropathic pain.     

Effects of TrkA inhibitory peptide on cancer-induced pain in a mouse melanoma model

Purpose

Tropomyosin receptor kinase (Trk) A, a high-affinity receptor of nerve growth factor, is a therapeutic target for both noxious and neuropathic pain. The present study examined the effects of an inhibitory peptide of Trk activity (IPTRK) 3 that inhibits TrkA activity on cancer-induced pain in a mouse melanoma model.

Methods

The hind paws of mice were inoculated with B16-F1 mouse melanoma cells on day 0. We administered IPTRK3 (20?mg/kg i.p.) repetitively on days 5, 6, 7, 8, and 9, and evaluated pain-related behaviors on days 0, 5, 10, 15, and 20 after tumor inoculation.

Results

Following inoculation, mice demonstrated mechanical allodynia and thermal hyperalgesia with an increased number of flinches, and paw volume increased gradually. However, an intraperitoneal injection of IPTRK3 significantly inhibited mechanical allodynia on day 15 and suppressed the number of flinches on day 20. The increased paw volume was significantly suppressed on day 20 after tumor inoculation. IPTRK3, however, showed no significant effect on thermal hyperalgesia.

Conclusions

These results suggest that TrkA inhibitory peptide likely suppress melanoma-induced pain with concomitant reduction in the increased paw volume in a mouse skin cancer pain model.     

The effect of systemic zonisamide (Zonegran) on thermal hyperalgesia and mechanical allodynia in rats with an experimental mononeuropathy

We studied the ability of zonisamide (Zonegran) to relieve thermal hyperalgesia and/or mechanical allodynia in the chronic constriction injury model of neuropathic pain. Zonisamide (25, 50, or 100 mg/kg) or saline was administered in a blinded, randomized manner by intraperitoneal injection on postoperative days (PODs) 4, 5, and 6. Paw withdrawal latency (PWL) to heat, paw withdrawal response to von Frey monofilaments, and pain scores based on weight-bearing were tested: before surgery; before and after zonisamide or saline (PODs 4, 5, and 6); and on POD 9. Systemic zonisamide relieved thermal hyperalgesia in a dose-dependent manner. All PWLs were significantly increased after zonisamide administration compared with pre-zonisamide measurements, except with the 100 mg/kg dose on POD 5. After zonisamide 100 mg/kg administration, there was a sustained increase in PWL on PODs 5 and 9, with significant carryover effect from the previous dose. However, zonisamide had little effect on mechanical allodynia, except at the 100 mg/kg dose, which was sedating in the rat. At the 100 mg/kg dose, paw withdrawal response was increased on PODs 4 and 5, whereas pain scores were reduced on PODs 4, 5, and 6. Pain scores were inconsistently reduced after 50 mg/kg or 25 mg/kg doses. IMPLICATIONS: Zonisamide causes a dose-related decrease in heat sensitivity in a rat model of neuropathic pain, but relieves mechanical sensitivity only in a dose that is sedating to the rat. Zonisamide may be useful in the treatment of some types of neuropathic pain.