Prolonged nociceptive responses to hind paw formalin injection in rats with a spinal cord injury.

Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS- and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.

Expansion of formalin-evoked Fos-immunoreactivity in rats with a spinal cord injury.

Peripheral tissue injury as well as spinal cord injury (SCI) may lead to sensitization of dorsal horn neurons and alterations in nociceptive processing. Thus, peripheral injuries experienced by SCI patients, even if not initially perceived, could result in a persistent and widespread activation of dorsal horn neurons and emerge as chronic pain with interventive repair or modest recovery from SCI. To visualize the spinal neuron response to peripheral tissue injury following complete SCI in rats, the neural transcription factor Fos was quantitated in the spinal cord. Two weeks following either a complete transection of the spinal cord at the level of T8 or a sham surgery (laminectomy), rats were injected with formalin into the left hind paw. Sham-operated rats demonstrated biphasic hind paw pain-related behavior following formalin injection, but transected rats displayed fewer behaviors in the second (tonic) phase. Stereological analysis of the sham group revealed that the extent of formalin-induced Fos expression was within the lumbar dorsal horn, with numerous Fos-like immunoreactive profiles in the ipsilateral dorsal horn and some contralateral immunoreactive profiles. In contrast, the level of Fos-like immunoreactivity in the transected group was significantly elevated and expanded in range compared to the sham group, with increases observed in the normal laminar distribution regions, as well as multi-segmentally through sacral levels and increases in the contralateral dorsal horn segments. The data demonstrate that widespread activation of spinal, especially dorsal horn, neurons following peripheral insult can occur in the injured spinal cord, despite reduced pain responsiveness, and suggests that exaggerated pain may emerge as spinal recovery or repair progresses.

Antinociceptive effects of topical mepivacaine in a rat model of HIV-associated peripheral neuropathic pain.

BACKGROUND: A consequence of HIV infection is sensory neuropathy, a debilitating condition that degrades the quality of life of HIV patients. Furthermore, life-extending antiretroviral treatment may exacerbate HIV sensory neuropathy. Analgesics that relieve other neuropathic pains show little or no efficacy in ameliorating HIV sensory neuropathy. Thus, there is a need for analgesics for people with this particular pain. While lidocaine is used in the management of painful peripheral neuropathies, another local anesthetic mepivacaine, with a potentially improved bioavailability, could be utilized for the management of HIV neuropathic pain. METHODS: The efficacy of topical anesthetics was evaluated in a preclinical rodent model of painful peripheral neuropathy induced by epineural administration of the HIV envelope protein gp120 delivered using saturated oxidized cellulose implanted around the sciatic nerve. Beginning at 2 weeks following gp120 administration, the effects of local anesthetics topically applied via gauze pads were tested on heat and mechanical hyperalgesia in the hind paw. Rats were tested using several concentrations of mepivacaine or lidocaine during the following 2 weeks. RESULTS: By 2 weeks following epineural gp120 implantation, the ipsilateral hind paw developed significant hypersensitivity to noxious pressure and heat hyperalgesia. A short-lasting, concentration-dependent amelioration of pressure and heat hyperalgesia was observed following topical application of mepivacaine to the ipsilateral plantar hind paw. By contrast, topical lidocaine ameliorated heat hyperalgesia in a concentration-dependent manner but not pressure hyperalgesia. Equipotent concentrations of mepivacaine and lidocaine applied topically to the tail of mice significantly increased tail withdrawal latencies in the tail flick test, demonstrating that both local anesthetics attenuate responding to a brief noxious stimulus. CONCLUSION: These findings showed that mepivacaine, rather than lidocaine, consistently attenuated two distinct symptoms of neuropathic pain and suggest that topical formulations of this local anesthetic could have utility in the alleviation of clinical HIV neuropathic pain.

Behavioral and pharmacological characterization of a distal peripheral nerve injury in the rat.

Previous rat neuropathic pain models have utilized peripheral nerve injuries that damage a significant proportion of large nerves such as the sciatic nerve or its divisions. Injuries that lead to neuropathic pain in humans may involve the peripheral extremities. The current study evaluated the behavioral effects of injury to the plantar nerves in the rat (distal nerve injury-DNI). A delayed onset of hypersensitivity to an innocuous mechanical stimulus was observed following cutting of the left plantar nerves, whereas mechanical hypersensitivity developed more rapidly in rats with either an injury near the sciatic nerve trunk (chronic constriction injury (CCI), spared nerve injury (SNI)) or a spinal nerve root (spinal nerve ligation (SNL). Similar to other nerve injury pain models, rats with injured plantar nerves also developed an early onset and persistent sensitivity to a cooling stimulus. The effects of morphine, gabapentin and imipramine on mechanical and cold hypersensitivity were evaluated in rats with a DNI, CCI and SNI. In all three models, morphine dose-dependently suppressed mechanical and cold hypersensitivity, whereas gabapentin only suppressed mechanical hypersensitivity. Imipramine had no effect on either cold or mechanical hypersensitivity in any of the nerve-injured rats. The pharmacological data suggest that the underlying basis of neuropathic pain may be similar irrespective of the site of nerve injury.

Reduced pain-related behavior by adrenal medullary transplants in rats with experimental painful peripheral neuropathy.

Adrenal medullary transplants in the spinal subarachnoid space, by providing a continual source of opioid peptides and catecholamines, offer a potentially important adjunct in the management of chronic pain. While previous studies have shown that this approach is effective against high-intensity phasic stimuli, adrenal medullary implants need to be evaluated against long-term and abnormal pain syndromes before transplantation can be used for human chronic pain. Using a recently developed model of painful peripheral neuropathy, the effects of adrenal medullary chromaffin cells transplanted into the subarachnoid space was evaluated. Peripheral mononeuropathy was induced by loosely tying 4 ligatures (4-0 chromic gut) around the right sciatic nerve. This procedure produces various pain symptoms including allodynia, hyperalgesia and dysesthesia. Rats were given either adrenal medullary tissue or control striated muscle transplants. Animals with adrenal medullary tissue transplants showed markedly decreased allodynia to innocuous cold as early as 1 week post-transplantation. In addition, hyperalgesia to a noxious thermal stimulus was eliminated by adrenal medullary, but not control, transplants. Touch-evoked allodynia was only slightly reduced by adrenal medullary transplants. In addition, indicators of spontaneous pain appeared reduced in animals with adrenal medullary transplants. These findings indicate that adrenal medullary transplants may be effective in reducing neuropathic pain.

Acute activation of the spinal cord metabotropic glutamate subtype-5 receptor leads to cold hypersensitivity in the rat.

Activation of spinal cord dorsal horn ionotropic glutamate receptors leads to pain-related behaviors. However, the role of spinal metabotropic glutamate receptors (mGlu), particularly the mGlu5 receptor subtype, in nociception has not been well characterized. A recently described subtype selective and potent mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP) was used to evaluate the role of the mGlu5 receptor in cold sensitivity. Intrathecal (i.t.) injection of group I (mGlu1 and mGlu5 receptors) mGlu receptor-selective agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) increased the hind paw frequency and duration of lifting of rats placed on a cold (4 degrees C) surface, a behavior similarly observed in rats with a chronic constriction injury (CCI) of the sciatic nerve. In contrast, rats i.t. injected with DHPG did not display increased lifting when placed on a room temperature surface. I.t. injection of MPEP before i.t. injection of DHPG blocked DHPG-evoked cold hypersensitivity, suggesting that activation of spinal mGlu5 receptors induces this behavioral response. In contrast, i.t. injection of MPEP after i.t. injection of DHPG had no effect. In addition, i.t. injection of MPEP did not affect cold hypersensitivity in rats with a CCI. These data suggest that acute activation of spinal cord mGlu5 receptors results in increased sensitivity to cold, but ongoing cold hypersensitivity does not involve activation of the mGlu5 receptor.

Enhanced antinociception by nicotinic receptor agonist epibatidine and adrenal medullary transplants in the spinal subarachnoid space.

Adrenal medullary transplants in the spinal subarachnoid space can reduce nociception, via the release of catecholamines and other analgesic substances, and this may be enhanced by stimulation of transplanted chromaffin cell surface nicotinic acetylcholine receptors (nAChRs). In addition, spinal nAChRs have been implicated in modulating nociception and can interact synergistically with alpha-adrenergic agents. Thus, enhanced antinociception by potent nAChR agonists such as frog skin derivative epibatidine in adrenal-transplanted animals could potentially occur via multiple mechanisms, including nicotinic-alpha-adrenergic synergy and stimulation of chromaffin cell nicotinic receptors. In order to test this, male Sprague-Dawley rats were implanted with intrathecal catheters and either adrenal medullary or control striated muscle transplants in the spinal subarachnoid space at the lumbar enlargement. Animals were tested for nociceptive responses before and after intrathecal injection of several doses of epibatidine using acute analgesiometric tests (tail flick, paw pressure) and the formalin test. After adrenal medullary, but not control, transplantation, nociceptive thresholds to acute noxious stimuli were slightly but consistently elevated, and phase 2 formalin responses decreased. Following intrathecal injection of epibatidine, acute nociceptive response latencies were modestly elevated and phase 2 formalin flinches modestly suppressed in control animals, but only at the highest dose test, with some attendant motor side-effects. In contrast, in adrenal medullary-transplanted animals, epibatidine elevated responses to acute noxious stimuli and markedly suppressed phase 2 formalin responses in a dose-related fashion. The enhanced antinociceptive effect following epibatidine was attenuated with either nAChR antagonist mecamylamine or alpha-adrenergic receptor antagonist phentolamine. The current results demonstrate that intrathecal injection of the nAChR ligand epibatidine can produce significant antinociception in adrenal-transplanted rats in both acute and tonic nociceptive tests and suggest that the use of nicotinic agents in combination with adrenal medullary transplantation could provide maximal therapeutic benefit by synergistically improving antinociception while avoiding the detrimental side-effects of these agents.

Capsaicin-sensitive primary afferents mediate responses to cold in rats with a peripheral mononeuropathy.

Persistent sensitivity to noxious and innocuous somatic stimuli results from peripheral nerve injury. Following chronic constriction injury (CCI) of the sciatic nerve in the rat, the hind paw ipsilateral to the injury displays significantly decreased response latencies to a noxious heat stimulus (thermal hyperalgesia), compared with the contralateral uninjured paw. The ligated paw also shows increased lifting and duration of lifting from a cooled (4 +/- 1 degrees C) surface. To characterize the peripheral nerve component of increased sensitivity to cold, CCI rats were systemically injected with the potent capsaicin analog resiniferatoxin (RTX). Twenty-four hours following RTX injection response latencies to noxious heat were significantly increased for both the ligated and unligated hind paws. In addition, increased responsiveness of the ligated paw to the cold surface was significantly attenuated. The results demonstrate that the enhanced responsiveness to cold and heat following a CCI are mediated in part by RTX-sensitive primary afferents.

Antihyperalgesic effect of the cannabinoid agonist WIN55,212-2 is mediated through an interaction with spinal metabotropic glutamate-5 receptors in rats.

In the current study, a possible interaction between spinal cord dorsal horn cannabinoid and mGlu5 receptors was evaluated in rats with a peripheral nerve injury. Following unilateral loose ligation of a sciatic nerve, rats developed decreased withdrawal thresholds to noxious pressure (mechanical hyperalgesia) of the ligated but not the unoperated contralateral hind paw. Systemic (subcutaneous) injection of synthetic cannabinoid agonist WIN55,212-2 increased withdrawal thresholds of both the ligated and the unoperated hind paw. Systemic injection of 2-methyl-6-(phenylethynyl)pyradine (MPEP), an antagonist selective for the mGlu5 receptor, did not alter the antinociceptive and antihyperalgesic effects of systemic WIN55,212-2. Intrathecal (i.t.) injection of WIN55,212-2 increased thresholds of the ligated but not the unoperated hind paw. Intrathecal injection of MPEP reversed the antihyperalgesic effect of i.t. WIN55,212-2. Neither systemic nor i.t. injection of MPEP alone altered withdrawal thresholds. These data suggest that the antihyperalgesic effect of WIN55,212-2 is mediated through an interaction with spinal mGlu5 receptors.

Effects of repeated dosing with mechanistically distinct antinociceptive ligands in a rat model of neuropathic spinal cord injury pain.

A lack of efficacy of some analgesic drugs has been previously described in rats with neuropathic spinal cord injury (SCI) pain. It has been suggested that repeated dosing in these animals over time may eventually lead to efficacy. However, it is also possible that efficacy may diminish over time with repeated dosing. This study evaluated the efficacy of various drugs upon repeated dosing over time in a rat model of SCI pain. Four weeks following an acute spinal cord compression at the mid-thoracic level, rats developed decreased hind paw withdrawal threshold, suggestive of below level neuropathic hypersensitivity. Either cannabinoid (CB) receptor agonist CP 55,940, the anticonvulsant carbamazepine or gabapentin, the antidepressant amitriptyline or vehicle was administered over a period of 7 days. Neither carbamazepine nor amitriptyline demonstrated efficacy either after a single or repeated dosing. Beginning with a 50% efficacious dose of gabapentin, the effect of gabapentin in SCI rats neither increased nor decreased over the treatment period. The antinociceptive effect of CP 55,940 was maintained for the entire treatment period, which was mediated by CB1 but not CB2 receptors. The current data suggest that sustained antinociception can be obtained with some drugs in rats with neuropathic SCI pain. Furthermore, the current data do not substantiate the notion that repeated treatment with initially ineffective drugs will eventually lead to efficacy; treatments that are not acutely effective are unlikely to demonstrate clinical efficacy.

Fatty acid amide hydrolase (FAAH) inhibitors exert pharmacological effects, but lack antinociceptive efficacy in rats with neuropathic spinal cord injury pain.

Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

Beyond rodent models of pain: non-human primate models for evaluating novel analgesic therapeutics and elaborating pain mechanisms.

Evaluation of potential analgesic therapeutics and the elaboration of the neurobiology of pain have heavily relied on pain models developed in rodents. However, a limitation of rodents is their phylogenetic distance from humans, which could in part account for the failure of some preclinical findings to translate to clinical utility. By contrast, given their genetic closeness and phenotypic similarities to humans, it is suggested that there be greater utilization of non-human primates (NHP) in preclinical pain studies. Methods to induce chronic pain-like states and quantify changes in nociception that have been developed in rodents could be adapted to the NHP. Similarly, human experimental injury-induced sensitization, which attempts to temporarily mimic the neuropathology and symptoms observed in the chronic pain state, could be adapted to the NHP. The NHP could then serve as a platform to validate human experimental models as well as proof-of-concept studies. Beyond experimentally modeled pain states, a number of naturally occurring disease states, such as osteoarthritis, are expressed by NHP, which could be utilized for both hypothesis testing and proof-of-concept studies. While NHP studies are logistically cumbersome, it is envisioned that NHP pain models will add value to current preclinical data and greatly facilitate the discovery of novel analgesic treatments.

The antinociceptive effect of the asthma drug ibudilast in rat models of peripheral and central neuropathic pain.

Ibudilast, an asthma drug, has demonstrated antinociceptive effects in several rat models of peripheral neuropathic pain, and a proposed mechanism of action is the inhibition of release of the cytokine tumor necrosis factor-α (TNF-α) from activated spinal microglia. Spinal glial activation has also been demonstrated in rat models of central neuropathic pain following spinal cord injury (SCI). The current study evaluated the effect of a short course of treatment with ibudilast on SCI-induced pain, and for comparison, following a chronic constriction injury (CCI; the Bennett model) of the sciatic nerve in rats. The effects of ibudilast treatment on spinal (SCI and CCI rats), and nerve tissue (CCI only) TNF-α content were also evaluated. Following an acute midthoracic SCI with a microvascular clip (20-g force), hindpaw withdrawal thresholds were significantly decreased, indicating below-level cutaneous tactile hypersensitivity. Likewise, unilateral loose ligation of the sciatic nerve led to a robust ipsilateral tactile hypersensitivity. Rats were treated with either ibudilast (10 mg/kg IP) or vehicle (2 mL/kg) during the period of robust and steady hindpaw hypersensitivity for each model--CCI rats were treated 14-16 days post-surgery, and SCI rats were treated 30-32 days post-surgery--and tested daily. Ibudilast ameliorated hindpaw hypersensitivity in both SCI and CCI rats, whereas vehicle treatment had no effect. Interestingly, repeated treatment led to increased baseline thresholds, beyond the duration of the drug half-life, suggesting persistent changes in neuropathic pain processing. In SCI rats, an increase in TNF-α content in spinal tissue rostral to the SCI was observed. Ibudilast treatment did not significantly alter this increase. In rats with a CCI, TNF-α content was markedly increased in the ipsilateral sciatic nerve and was partially reduced following ibudilast, but not vehicle, treatment. Ibudilast could be useful for the treatment of neuropathic pain of central as well as peripheral origin.

Cannabinoid receptor-mediated antinociception with acetaminophen drug combinations in rats with neuropathic spinal cord injury pain.

Pre-clinical evidence demonstrates that neuropathic spinal cord injury (SCI) pain is maintained by a number of neurobiological mechanisms, suggesting that treatments directed at several pain-related targets may be more advantageous compared to a treatment focused on a single target. The current study evaluated the efficacy of the non-opiate analgesic acetaminophen, which has several putative analgesic mechanisms, combined with analgesic drugs used to treat neuropathic pain in a rat model of below-level neuropathic SCI pain. Following an acute compression of the mid-thoracic spinal cord, rats exhibited robust hind paw hypersensitivity to innocuous mechanical stimulation. Fifty percent antinociceptive doses of gabapentin, morphine, tramadol or memantine were combined with an ineffective dose of acetaminophen; acetaminophen alone was not antinociceptive. The combination of acetaminophen with either tramadol or memantine resulted in an additive antinociceptive effect. Acetaminophen combined with either morphine or gabapentin, however, resulted in supra-additive (synergistic) efficacy. One of the analgesic mechanisms of acetaminophen is inhibiting the uptake of endocannabinoids from the extracellular space. Pre-treatment with AM251, a cannabinoid-1 receptor (CB1) antagonist, significantly diminished the antinociceptive effect of the acetaminophen + gabapentin combination. Pre-treatment with AM630, a cannabinoid-2 receptor (CB2) antagonist, did not have an effect on this combination. By contrast, both AM251 and AM630 reduced the efficacy of the acetaminophen + morphine combination. None of the active drugs alone were affected by either CB receptor antagonist. The results imply that modulation of the endocannabinoid system in addition to other mechanisms mediate the synergistic antinociceptive effects of acetaminophen combinations. Despite the presence of a cannabinoid mechanism, synergism was not present in all acetaminophen combinations. The combination of currently available drugs may be an appropriate option in ameliorating neuropathic SCI pain if single drug therapy is ineffective.

Antinociceptive effect of ambroxol in rats with neuropathic spinal cord injury pain.

Symptoms of neuropathic spinal cord injury (SCI) pain include evoked cutaneous hypersensitivity and spontaneous pain, which can be present below the level of the injury. Adverse side-effects obtained with currently available analgesics complicate effective pain management in SCI patients. Voltage-gated Na(+) channels expressed in primary afferent nociceptors have been identified to mediate persistent hyperexcitability in dorsal root ganglia (DRG) neurons, which in part underlies the symptoms of nerve injury-induced pain. Ambroxol has previously demonstrated antinociceptive effects in rat chronic pain models and has also shown to potently block Na(+) channel current in DRG neurons. Ambroxol was tested in rats that underwent a mid-thoracic spinal cord compression injury. Injured rats demonstrated robust hind paw (below-level) heat and mechanical hypersensitivity. Orally administered ambroxol significantly attenuated below-level hypersensitivity at doses that did not affect performance on the rotarod test. Intrathecal injection of ambroxol did not ameliorate below-level hypersensitivity. The current data suggest that ambroxol could be effective for clinical neuropathic SCI pain. Furthermore, the data suggest that peripherally expressed Na(+) channels could lend themselves as targets for the development of pharmacotherapies for SCI pain.

The effect of antinociceptive drugs tested at different times after nerve injury in rats.

Given the evolving nature of anatomical and functional changes in the nervous system that are involved in the development of neuropathic pain, it is possible that the differing time course after injury underlies the inconsistent efficacy of drugs in neuropathic pain patients. In the current study, we evaluated the behavioral effects of two standard drugs used clinically for neuropathic pain, the anticonvulsant gabapentin and antidepressant imipramine, in rats at different times after peripheral nerve injury. Rats that underwent the spared nerve injury procedure responded to an innocuous mechanical stimulus (von Frey filament) 2, 4, and 8 wk after injury. Gabapentin dose-dependently suppressed mechanical sensitivity at all time points tested but the potency of gabapentin was three-fold less 4 wk postinjury (135 mg/kg) compared with 2 and 8 wk postinjury (41 and 44 mg/kg, respectively). In contrast, imipramine lacked significant efficacy at 2 and 8 wk postinjury but slightly attenuated mechanical hypersensitivity at 4 wk postinjury. The results show that drug effects may change over time in the neuropathic state, which should be an important consideration in the evaluation of drugs in preclinical animal pain models and has implications for temporal approaches to therapy in the clinic.