Josephine Lai

Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS.

We designed AM1241, a selective CB2 cannabinoid receptor agonist, and used it to test the hypothesis that CB2 receptor activation would reverse the sensory hypersensitivity observed in neuropathic pain states. AM1241 exhibits high affinity and selectivity for CB2 receptors. It also exhibits high potency in vivo. AM1241 dose-dependently reversed tactile and thermal hypersensitivity produced by ligation of the L5 and L6 spinal nerves in rats. These effects were selectively antagonized by a CB2 but not by a CB1 receptor antagonist, suggesting that they were produced by actions of AM1241 at CB2 receptors. AM1241 was also active in blocking spinal nerve ligation-induced tactile and thermal hypersensitivity in mice lacking CB1 receptors (CB1-/- mice), confirming that AM1241 reverses sensory hypersensitivity independent of actions at CB1 receptors. These findings demonstrate a mechanism leading to the inhibition of pain, one that targets receptors localized exclusively outside the CNS. Further, they suggest the potential use of CB2 receptor-selective agonists for treatment of human neuropathic pain, a condition currently without consistently effective therapies. CB2 receptor-selective agonist medications are predicted to be without the CNS side effects that limit the effectiveness of currently available medications.

Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8.

&NA; Neuropathic pain is a debilitating chronic syndrome that often arises from injuries to peripheral nerves. Such pain has been hypothesized to be the result of an aberrant expression and function of sodium channels at the site of injury. Here, we show that intrathecal administration of specific antisense oligodeoxynucleotides (ODN) to the peripheral tetrodotoxin (TTX)‐resistant sodium channel, NaV1.8, resulted in a time‐dependent uptake of the ODN by dorsal root ganglion (DRG) neurons, a selective ‘knock‐down’ of the expression of NaV1.8, and a reduction in the slow‐inactivating, TTX‐resistant sodium current in the DRG cells. The ODN treatment also reversed neuropathic pain induced by spinal nerve injury, without affecting non‐noxious sensation or response to acute pain. These data provide direct evidence linking NaV1.8 to neuropathic pain. As NaV1.8 expression is restricted to sensory neurons, this channel offers a highly specific and effective molecular target for the treatment of neuropathic pain.

Unmasking the tonic-aversive state in neuropathic pain

Tonic pain has been difficult to demonstrate in animals. Because relief of pain is rewarding, analgesic agents that are not rewarding in the absence of pain should become rewarding only when there is ongoing pain. We used conditioned place preference to concomitantly determine the presence of tonic pain in rats and the efficacy of agents that relieve it. This provides a new approach for investigating tonic pain in animals and for evaluating the analgesic effects of drugs.

Distinct potassium channels on pain-sensing neurons

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K+ (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 α subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in Aδ and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na+ channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 α subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.

Spinal and Supraspinal Mechanisms of Neuropathic Pain

Abstract: Neuropathic pain is associated with abnormal tactile and thermal responses that may be extraterritorial to the injured nerve. Importantly, tactile allodynia and thermal hyperalgesia may involve separate pathways, since complete and partial spinal cord lesions have blocked allodynia, but not hyperalgesia, after spinal nerve ligation (SNL). Furthermore, lesions of the dorsal column, and lidocaine microinjected into dorsal column nuclei block only tactile allodynia. Conversely, thermal hyperalgesia, but not tactile allodynia was blocked by desensitizatin of C‐fibers with resiniferotoxin. Therefore, it seems that tactile allodynia is likely to be mediated by large diameter Aβ fibers, and not susceptible to modulation by spinal opioids, whereas hyperalgesia is mediated by unmyelinated C‐fibers, and is sensitive to blockade by spinal opioids. Additionally, abnormal, spontaneous afferent drive in neuropathic pain may contribute to NMDA‐mediated central sensitization by glutamate and by non‐opioid actions of spinal dynorphin. Correspondingly, SNL elicited elevation in spinal dynorphin content in spinal segments at and adjacent to the zone of entry of the injured nerve along with signs of neuropathic pain. Antiserum to dynorphin A(1–17) or MK‐801 given spinally blocked thermal hyperalgesia, but not tactile allodynia, after SNL, and also restored diminished morphine antinociception. Finally, afferent drive may induce descending facilitation from the rostroventromedial medulla (RVM). Blocking afferent drive with bupivicaine also restored lost potency of PAG morphine, as did CCK antagonists in the RVM. This observation is consistent with afferent drive activating descending facilitation from the RVM, and thus diminishing opioid activity, and may underlie the clinical observation of limited responsiveness of neuropathic pain to opioids

Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats

Tactile allodynia and thermal hyperalgesia, two robust signs of neuropathic pain associated with experimental nerve injury, have been hypothesized to be mechanistically distinguished based on (a) fiber types which may be involved in the afferent input, (b) participation of spinal and supraspinal circuitry in these responses, and (c) sensitivity of these endpoints to pharmacological agents. Here, the possibility that nerve-injury induced tactile allodynia and thermal hyperalgesia may be mediated via different afferent fiber input was tested by evaluating these responses in sham-operated or nerve-injured (L5/L6) rats before or after a single systemic injection of resiniferatoxin (RTX), an ultrapotent analogue of the C-fiber specific neurotoxin, capsaicin. Tactile allodynia, and three measures of thermal nociception, tail-flick, paw-flick and hot-plate responses, were determined before and at various intervals for at least 40 days after RTX injection. Nerve-injured, but not sham-operated, rats showed a long-lasting tactile allodynia and thermal hyperalgesia (paw-flick) within 2-3 days after surgery; responses to other noxious thermal stimuli (i.e., tail-flick and hot-plate tests) did not distinguish the two groups at the stimulus intensities employed. RTX treatment resulted in a significant and long-lasting (i.e. essentially irreversible) decrease in sensitivity to thermal noxious stimuli in both sham-operated and nerve-injured rats; thermal hyperalgesia was abolished and antinociception produced by RTX. In contrast, RTX treatment did not affect the tactile allodynia seen in the same nerve-injured rats. These data support the concept that thermal hyperalgesia seen after nerve ligation, as well as noxious thermal stimuli, are likely to be mediated by capsaicin-sensitive C-fiber afferents. In contrast, nerve-injury related tactile allodynia is insensitive to RTX treatment which clearly desensitizes C-fibers and, therefore such responses are not likely to be mediated through C-fiber afferents. The hypothesis that tactile allodynia may be due to inputs from large (i.e. A beta) afferents offers a mechanistic basis for the observed insensitivity of this endpoint to intrathecal morphine in this nerve-injury model. Further, these data suggest that clinical treatment of neuropathic pains with C-fiber specific agents such as capsaicin are unlikely to offer significant therapeutic benefit against mechanical allodynia.

Underlying Mechanisms of Pronociceptive Consequences of Prolonged Morphine Exposure

The opioid analgesics, commonly exemplified by morphine, represent the best option for the treatment of severe pain and for the management of chronic pain states, of both malignant and nonmalignant origin. It is well recognized that the prolonged use of opioids is associated with a requirement for ever‐increasing doses in order to maintain pain relief at an acceptable and consistent level. This phenomenon is termed analgesic tolerance. While the concept that tolerance can develop as a result of cellular adaptations to the presence of the opioid has been proposed, it is now becoming abundantly clear that tolerance may also be related to a state of hyperalgesia that results from exposure to the opioid itself. Patients who receive long‐term opioid therapy sometimes develop unexpected, abnormal pain. Similar paradoxical opioid‐induced pain has been confirmed in a number of animal studies, even during the period of continuous opioid delivery. A number of recent studies have demonstrated that such pain may be secondary to neuroplastic changes that occur in the brain and spinal cord. One such change may be the activation of descending pain facilitation mechanisms arising from the rostral ventromedial medulla (RVM) elicited in part by increased activity of cholecystokinin (CCK) in the RVM. A cascade of pronociceptive events may follow, such as opioid‐induced upregulation of spinal dynorphin levels that promotes enhanced input from primary afferent nociceptors. This mechanism appears to depend on intact descending pathways from the RVM, since interrupting this pathway abolishes enhanced abnormal pain. Furthermore, extended opioid exposure also can elicit increased calcitonin gene related peptide (CGRP) and substance P expression in the dorsal root ganglia. It is probable that increased pain elicited by opioids is a critical factor in the behavioral manifestation of opioid tolerance because the same manipulations that block abnormal pain also block antinociceptive tolerance. Taken together, such studies show that opioids elicit systems‐level adaptations resulting in pain due to descending facilitation, upregulation of spinal dynorphin, and enhanced, evoked release of excitatory transmitters from primary afferents. These adaptive changes in response to sustained exposure to opioids indicate the need for the evaluation of the clinical consequences of long‐term opioid administration. Additionally, these findings suggest a need for novel chemistry involving design of agents that may counteract opiate‐induced neuroplastic adaptations resulting in pain relief without analgesic tolerance.

Reversal of experimental neuropathic pain by T-type calcium channel blockers

Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca2+ current is associated with increased neuronal activity and blockade of N‐ and P‐types, but not L‐type, calcium channels have been found to block experimental neuropathic pain. While T‐type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L5/L6 spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose‐dependent blockade of both tactile and thermal hypersensitivities in nerve‐injured rats; responses of sham‐operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T‐type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T‐type calcium channel blockers may have significant potential in the treatment of neuropathic pain states.

Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat.

&NA; Complete or partial spinal section at T8 has been shown to block tactile allodynia but not thermal hyperalgesia following L5/L6 spinal nerve ligation (SNL), suggesting the supraspinal integration of allodynia in neuropathic pain. In the present study, the possibility of mediation of nerve injury‐associated pain through tonic activity of descending nociceptive facilitation arising from the rostroventromedial medulla (RVM) was investigated. Specifically, the actions of brainstem cholecystokinin and the possible importance of sustained afferent input from injured nerve fibers were determined using pharmacological and physiological approaches in rats with SNL. Lidocaine given bilaterally into the RVM blocked tactile allodynia and thermal hyperalgesia in SNL rats and was inactive in sham‐operated rats. Bilateral injection of L365,260 (CCKB receptor antagonist) into the RVM also reversed both tactile allodynia and thermal hyperalgesia. Microinjection of CCK‐8 (s) into the RVM of naive rats produced a robust tactile allodynic effect and a more modest hyperalgesia. CCK immunoreactivity was not significantly different between SNL and sham‐operated rats. The anti‐nociceptive effect of morphine given into the ventrolateral periaqueductal gray region (PAG) was substantially reduced by SNL. The injection of L365,260 into the RVM or of bupivacaine at the site of nerve injury restored the potency and efficacy of PAG morphine in SNL rats. These results suggest that changes in supraspinal processing are likely to contribute to the observed poor efficacy of opioids in clinical states of neuropathic pain. These data also indicate that the activation of descending nociceptive facilitatory pathways is important in the maintenance of neuropathic pain, appears to be dependent on CCK release, and may be driven from sustained afferent input from injured nerves to brainstem sites. Collectively, these data support the hypothesis that abnormal tonic activity of descending facilitation mechanisms may underlie chronic pain from peripheral nerve injury.

Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats

&NA; Neuropathic pain is often associated with the appearance of pain in regions not related to the injured nerve. One mechanism that may underlie neuropathic pain is abnormal, spontaneous afferent drive which may contribute to NMDA‐mediated central sensitization by the actions of glutamate and by the non‐opioid actions of spinal dynorphin. In the present study, injuries to lumbar or sacral spinal nerves elicited elevation in spinal dynorphin content which correlated temporally and spatially with signs of neuropathic pain. The increase in spinal dynorphin content was coincident with the onset of tactile allodynia and thermal hyperalgesia. Injury to the lumbar (L5/L6) spinal nerves produced elevated spinal dynorphin content in the ipsilateral dorsal spinal quadrant at the L5 and L6 spinal segments and in the segments immediately adjacent. Lumbar nerve injury elicited ipsilateral tactile allodynia and thermal hyperalgesia of the hindpaw. In contrast, S2 spinal nerve ligation elicited elevated dynorphin content in sacral spinal segments and bilaterally in the caudal lumbar spinal cord. The behavioral consequences of S2 spinal nerve ligation were also bilateral, with tactile allodynia and thermal hyperalgesia seen in both hindpaws. Application of lidocaine to the site of S2 ligation blocked thermal hyperalgesia and tactile allodynia of the hindpaws suggesting that afferent drive was critical to maintenance of the pain state. Spinal injection of antiserum to dynorphin A(1–17) and of MK‐801 both blocked thermal hyperalgesia, but not tactile allodynia, of the hindpaw after S2 ligation. These data suggest that the elevated spinal dynorphin content consequent to peripheral nerve injury may drive sensitization of the spinal cord, in part through dynorphin acting directly or indirectly on the NMDA receptor complex. Furthermore, extrasegmental increases in spinal dynorphin content may partly underlie the development of extraterritorial neuropathic pain.