Lucy A. Bee

Preclinical and early clinical investigations related to monoaminergic pain modulation

SummaryThe balance between descending controls, both excitatory and inhibitory, can be altered in various pain states. There is good evidence for a prominent α2-adrenoceptor-mediated inhibitory system and 5-HT3 (and likely also 5-HT2) serotonin receptor-mediated excitatory controls originating from brainstem and midbrain areas. The ability of cortical controls to influence spinal function allows for top-down processing through these monoamines. The links between pain and the comorbidities of sleep problems, anxiety, and depression may be due to the dual roles of noradrenaline and of 5-HT in these functions and also in pain. These controls appear, in the cases of peripheral neuropathy, spinal injury, and cancer-induced bone pain to be driven by altered peripheral and spinal neuronal processes; in opioid-induced hyperalgesia, however, the same changes occur without any pathophysiological peripheral process. Thus, in generalized pain states in which fatigue, mood changes, and diffuse pain occur, such as fibromyalgia and irritable bowel syndrome, one could suggest an abnormal engagement of descending facilitations with or without reduced inhibitions but with central origins. This would be an endogenous central malfunction of top-down processing, with the altered monoamine systems underlying the observed symptoms. A number of analgesic drugs can either interact with or have their actions modulated by these descending systems, reinforcing their importance in the establishment of pain but also in its control.

Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

To examine the role of small RNAs in peripheral pain pathways, we deleted the enzyme Dicer in mouse postmitotic damage-sensing neurons. We used a Nav1.8-Cre mouse to target those nociceptors important for inflammatory pain. The conditional null mice were healthy with a normal number of sensory neurons and normal acute pain thresholds. Behavioral studies showed that inflammatory pain was attenuated or abolished. Inflammatory mediators failed to enhance excitability of Nav1.8+ sensory neurons from null mutant mice. Acute noxious input into the dorsal horn of the spinal cord was apparently normal, but the increased input associated with inflammatory pain measured using c-Fos staining was diminished. Microarray and quantitative real-time reverse-transcription PCR (qRT-PCR) analysis showed that Dicer deletion lead to the upregulation of many broadly expressed mRNA transcripts in dorsal root ganglia. By contrast, nociceptor-associated mRNA transcripts (e.g., Nav1.8, P2xr3, and Runx-1) were downregulated, resulting in lower levels of protein and functional expression. qRT-PCR analysis also showed lowered levels of expression of nociceptor-specific pre-mRNA transcripts. MicroRNA microarray and deep sequencing identified known and novel nociceptor microRNAs in mouse Nav1.8+ sensory neurons that may regulate nociceptor gene expression.

Descending facilitation from the brainstem determines behavioural and neuronal hypersensitivity following nerve injury and efficacy of pregabalin

Abstract Various mechanisms at peripheral, spinal and/or supraspinal levels may underlie neuropathic pain. The nervous system’s capacity for long‐term reorganisation and chronic pain may result from abnormalities in RVM facilitatory On cells. Hence, via brainstem injections of the toxic conjugate dermorphin–saporin, which specifically lesions facilitatory cells expressing the μ‐opioid receptor (MOR), we sought to determine the influence of these cells in normal and spinal nerve‐ligated (SNL) rats. We combined behavioural, electrophysiological and pharmacological techniques to show that the supraspinal facilitatory drive is essential for neuronal processing of noxious stimuli in normal and neuropathic states, and that descending facilitatory neurones maintain behavioural hypersensitivities to mechanical stimuli during the late stages of nerve injury. Furthermore, we showed that these neurones are essential for the state‐dependent inhibitory actions of pregabalin (PGB), a drug used in the treatment of neuropathic pain. During the early stages of nerve injury, or following medullary MOR cell ablation, PGB is ineffective at inhibiting spinal neuronal responses possibly due to quiescent spinal 5HT3 receptors. This can however be overcome, and PGB’s efficacy restored, by pharmacologically mimicking the descending drive at the spinal level with a 5HT3 receptor agonist. Since RVM facilitatory neurones are integral to a spino‐bulbo‐spinal loop that reaches brain areas co‐ordinating the sensory and affective components of pain, we propose that activity therein may influence painful outcome following nerve injury, and responsiveness to treatment.

A role for Piezo2 in EPAC1-dependent mechanical allodynia

Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1−/− mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1–Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Mu-opioid and noradrenergic α2-adrenoceptor contributions to the effects of tapentadol on spinal electrophysiological measures of nociception in nerve-injured rats

&NA; Multiple pathological mechanisms at multiple sensory sites may underlie the pain that follows nerve injury. This provides a basis for recommending more than one agent, either sequentially or in combination, for its treatment. According to this premise, new drugs that combine different mechanisms of analgesic action in a single molecule are gaining momentum, such as tapentadol which stimulates mu‐opioid receptors (MOR) and acts as a noradrenaline reuptake inhibitor (NRI) in the CNS. Tapentadol is currently indicated for treating moderate to severe acute and severe chronic pain, and here we demonstrate its efficacy in an animal model of ongoing neuropathic pain. In particular, we performed a series of in vivo electrophysiological tests in spinal nerve ligated and sham‐operated rats to show that systemic tapentadol (1 and 5 mg/kg) dose‐dependently reduced evoked responses of spinal dorsal horn neurones to a range of peripheral stimuli, including brush, punctate mechanical and thermal stimuli. Furthermore, we showed that spinal application of the selective &agr;2‐adrenoceptor antagonist atipamezole, or alternatively the mu‐opioid receptor antagonist naloxone, produced near complete reversal of tapentadols inhibitory effects, which suggests not only that the spinal cord is the key site of tapentadols actions, but also that no pharmacology other than MOR‐NRI is involved in its analgesia. Moreover, according to the extent that the antagonists reversed tapentadols inhibitions in sham and SNL rats, we suggest that there may be a shift from predominant opioid inhibitory mechanisms in control animals, to predominant noradrenergic inhibition in neuropathic animals.

The Cav2.3 calcium channel antagonist SNX‐482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch‐evoked pain). Evidence exists for nerve damage‐induced plasticity in both transmission and modulatory systems, including changes in voltage‐dependent calcium channel (VDCC) expression and function; however, the role of Cav2.3 calcium channels has not clearly been defined. Here, the effects of SNX‐482, a selective Cav2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [ Kim & Chung, (1992)Pain, 50, 355–363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham‐operated animals. Spinal SNX‐482 (0.5–4 µg/50 µL) exerted dose‐related inhibitions of noxious C‐fibre‐ and Aδ‐fibre‐mediated neuronal responses in conditions of neuropathy, but not in sham‐operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX‐482. In contrast, non‐noxious Aβ‐mediated responses were not affected by SNX‐482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX‐482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX‐482‐sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in CaV2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.

A pronociceptive role for the 5-HT2 receptor on spinal nociceptive transmission: An in vivo electrophysiological study in the rat

Serotonin (5-HT) plays a major yet complex role in modulating spinal nociceptive transmission as a consequence of the number of 5-HT receptor subtypes. These include the 5-HT2 receptor, which is further sub classified into 5-HT2A, B and C. Studies have described both a pro- and antinociceptive action following 5-HT2A-receptor activation; therefore, to shed light on the directional nature of spinal 5-HT2A receptor activity, we investigated the effects of spinal administration of the 5-HT2A receptor antagonist, ketanserin, on the evoked responses of dorsal horn neurones to electrical, mechanical and thermal stimulation. We also assessed the effects of systemic administration of ritanserin, a 5-HT2A/2C receptor antagonist and spinal application of (±)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (DOI) (3.6 and 17.8 μg/50 μl), a 5-HT2A/2C agonist, on the same evoked neuronal responses. Ketanserin (1, 10 and 100 μg/50 μl) produced a dose related inhibition of the evoked responses to noxious mechanical punctate and thermal stimuli only. Ritanserin (2 mg/kg) replicated the inhibitory effects seen with ketanserin on the natural evoked neuronal responses and also potently inhibited the C-fibre, post discharge, input and wind-up evoked responses. DOI increased the mechanical and thermal evoked responses, an effect reversed by ketanserin. Thus, our findings show that spinal ketanserin (1–100 μg/50 μl) and systemic ritanserin (2 mg/kg), at these doses, have similar antinociceptive effects, whereas the agonist, DOI, produced excitatory effects, on spinal neuronal activity. Our data, therefore, supports a pronociceptive role for 5-HT2 receptors, most likely through modulation of 5-HT2A receptor activity, on spinal nociceptive transmission under normal conditions.

The lidocaine metabolite N-ethylglycine has antinociceptive effects in experimental inflammatory and neuropathic pain.

Abstract Glycine transporter 1 (GlyT1) plays a crucial role in regulating extracellular glycine concentrations and might thereby constitute a new drug target for the modulation of glycinergic inhibition in pain signaling. Consistent with this view, inhibition of GlyT1 has been found to induce antinociceptive effects in various animal pain models. We have shown previously that the lidocaine metabolite N-ethylglycine (EG) reduces GlyT1-dependent glycine uptake by functioning as an artificial substrate for this transporter. Here, we show that EG is specific for GlyT1 and that in rodent models of inflammatory and neuropathic pain, systemic treatment with EG results in an efficient amelioration of hyperalgesia and allodynia without affecting acute pain. There was no effect on motor coordination or the development of inflammatory edema. No adverse neurological effects were observed after repeated high-dose application of EG. EG concentrations both in blood and spinal fluid correlated with an increase of glycine concentration in spinal fluid. The time courses of the EG and glycine concentrations corresponded well with the antinociceptive effect. Additionally, we found that EG reduced the increase in neuronal firing of wide-dynamic-range neurons caused by inflammatory pain induction. These findings suggest that systemically applied lidocaine exerts antihyperalgesic effects through its metabolite EG in vivo, by enhancing spinal inhibition of pain processing through GlyT1 modulation and subsequent increase of glycine concentrations at glycinergic inhibitory synapses. EG and other substrates of GlyT1, therefore, may be a useful therapeutic agent in chronic pain states involving spinal disinhibition.

Synergistic Effect of 5-Hydroxytryptamine 3 and Neurokinin 1 Receptor Antagonism in Rodent Models of Somatic and Visceral Pain

Synergistic activity has been observed between serotonergic 5-hydroxytryptamine 3 (5-HT3) and tachykinergic neurokinin 1 (NK1) receptor–mediated responses. This study investigated the efficacy of a 5-HT3 antagonist, palonosetron, and a NK1 antagonist, netupitant, alone or in combination in rodent models of somatic and visceral colonic hypersensitivity. In a rat model of experimental neuropathic pain, somatic hypersensitivity was quantified by the number of ipsilateral paw withdrawals to a von Frey filament (6g). Electrophysiologic responses were recorded in the dorsal horn neurons after mechanical or thermal stimuli. Acute colonic hypersensitivity was induced experimentally in rats by infusing dilute acetic acid (0.6%) directly into the colon. Colonic sensitivity was assessed by a visceromotor behavioral response quantified as the number of abdominal contractions in response to graded isobaric pressures (0–60 mm Hg) of colorectal distension. Palonosetron or netupitant was administered alone or in combination via oral gavage. When dosed alone, both significantly reduced somatic sensitivity, decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation, and caused significant (P < 0.05) inhibition of colonic hypersensitivity in a dose-dependent manner. The combined administration of palonosetron and netupitant at doses that were ineffective alone significantly reduced both somatic and visceral sensitivity and decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation. In summary, the combination of palonosetron with a NK1 receptor antagonist showed synergistic analgesic activity in rodent models of somatic and visceral hypersensitivity, and may prove to be a useful therapeutic approach to treat pain associated with irritable bowel syndrome.

The importance of the descending monoamine system for the pain experience and its treatment.

Brainstem and midbrain areas engage descending facilitatory and inhibitory neurones to potentiate or suppress the passage of sensory inputs from spinal loci to the brain. The balance between descending controls, both excitatory and inhibitory, can be altered in various pain states and can critically determine the efficacy of certain analgesic drugs. There is good evidence for a prominent α2 adrenoceptor-mediated inhibitory system and for 5-HT3 receptor-mediated excitatory control of spinal cord activity that originates in supraspinal areas. Given the multiple roles of these transmitters in pain and functions such as sleep, depression, and anxiety, the link between spinal and supraspinal processing of noxious inputs (via the monoamine transmitters) could be pivotal for linking the sensory and affective components of pain and their common co-morbidities, and also may potentially explain differences in pain scores and treatment outcomes in the patient population.