Wahida Rahman

The Increased Trafficking of the Calcium Channel Subunit α2δ-1 to Presynaptic Terminals in Neuropathic Pain Is Inhibited by the α2δ Ligand Pregabalin

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit α2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both α2δ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of α2δ-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, α2δ-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of α2δ-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of α2δ-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of α2δ-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of α2δ-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury

Pain resulting from peripheral nerve injury, characterised by ongoing pain, hyperalgesia and allodynia arises from peripheral and central processes. Here, we studied the potential role of central facilitations in nerve injury by investigating the effect of blocking the excitatory 5HT3 receptor with ondansetron. 5HT3 receptors play a pronociceptive role in the spinal cord and ondansetron has previously been shown to produce antinociception in behavioural studies. We investigated the effects of spinally administered ondansetron (10, 50 and 100 microg) on the responses of deep dorsal horn neurones, evoked by peripheral electrical stimuli and a range of natural (mechanical punctate and heat) stimuli, 2 weeks after nerve injury induced through tight ligation of L5/6 spinal nerves (SNL). Comparisons were made between SNL rats and a sham-operated group. Ondansetron produced little effect on the electrically evoked responses (Abeta-, Adelta- and C-fibre-evoked responses, postdischarge); however, responses to mechanical punctate stimuli (von Frey filaments 1-75 g) were markedly reduced in both SNL and control groups. Furthermore, the drug effect was significantly enhanced after SNL (p<0.05). In particular, the lowest dose (10 microg) now became effective after SNL. Ondansetron produced less marked effects on thermal responses. Our results demonstrate that neuropathic pain states are associated with an enhanced descending facilitatory control of mechanical responses of spinal neurones, mediated through the activation of spinal 5HT3 receptors. These excitatory influences are likely to contribute to the development and maintenance of central sensitisation in the spinal cord, and furthermore, to the behavioural manifestation of tactile allodynia.

Spinal-supraspinal serotonergic circuits regulating neuropathic pain and its treatment with gabapentin

&NA; Not all neuropathic pain patients gain relief from current therapies that include the anticonvulsant, gabapentin, thought to modulate calcium channel function. We report a neural circuit that is permissive for the effectiveness of gabapentin. Substance P‐saporin (SP‐SAP) was used to selectively ablate superficial dorsal horn neurons expressing the neurokinin‐1 receptor for substance P. These neurons project to the brain as shown by retrograde labelling and engage descending brainstem serotonergic influences that enhance spinal excitability via a facilitatory action on 5HT3 receptors. We show the integrity of this pathway following nerve injury contributes to the behavioural allodynia, neuronal plasticity of deep dorsal horn neurons and the injury‐specific actions of gabapentin. Thus SP‐SAP attenuated the tactile and cold hypersensitivity and abnormal neuronal coding (including spontaneous activity, expansion of receptive field size) seen after spinal nerve ligation. Furthermore the powerful actions of gabapentin after neuropathy were blocked by either ablation of NK‐1 expressing neurones or 5HT3 receptor antagonism using ondansetron. Remarkably, 5HT3 receptor activation provided a state‐dependency (independent of that produced by neuropathy) allowing GBP to powerfully inhibit in normal uninjured animals. This circuit is therefore a crucial determinant of the abnormal neuronal and behavioural manifestations of neuropathy and importantly, the efficacy of gabapentin. As this spino‐bulbo‐spinal circuit contacts areas of the brain implicated in the affective components of pain, this loop may represent a route by which emotions can influence the degree of pain in a patient, as well as the effectiveness of the drug treatment. These hypotheses are testable in patients.

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinum Toxin A Endopeptidase Fragment and Erythrina cristagalli Lectin

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LHN/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits anin vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed usingin vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LHN/A or recombinant LHN/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LHN/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed withClostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitroactions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.

Descending serotonergic facilitation and the antinociceptive effects of pregabalin in a rat model of osteoarthritic pain

BackgroundDescending facilitation, from the brainstem, promotes spinal neuronal hyperexcitability and behavioural hypersensitivity in many chronic pain states. We have previously demonstrated enhanced descending facilitation onto dorsal horn neurones in a neuropathic pain model, and shown this to enable the analgesic effectiveness of gabapentin. Here we have tested if this hypothesis applies to other pain states by using a combination of approaches in a rat model of osteoarthritis (OA) to ascertain if 1) a role for descending 5HT mediated facilitation exists, and 2) if pregabalin (a newer analogue of gabapentin) is an effective antinociceptive agent in this model. Further, quantitative-PCR experiments were undertaken to analyse the α2δ-1 and 5-HT3A subunit mRNA levels in L3–6 DRG in order to assess whether changes in these molecular substrates have a bearing on the pharmacological effects of ondansetron and pregabalin in OA.ResultsOsteoarthritis was induced via intra-articular injection of monosodium iodoacetate (MIA) into the knee joint. Control animals were injected with 0.9% saline. Two weeks later in vivo electrophysiology was performed, comparing the effects of spinal ondansetron (10–100 μg/50 μl) or systemic pregabalin (0.3 – 10 mg/kg) on evoked responses of dorsal horn neurones to electrical, mechanical and thermal stimuli in MIA or control rats. In MIA rats, ondansetron significantly inhibited the evoked responses to both innocuous and noxious natural evoked neuronal responses, whereas only inhibition of noxious evoked responses was seen in controls. Pregabalin significantly inhibited neuronal responses in the MIA rats only; this effect was blocked by a pre-administration of spinal ondansetron. Analysis of α2δ-1 and 5-HT3A subunit mRNA levels in L3–6 DRG revealed a significant increase in α2δ-1 levels in ipsilateral L3&4 DRG in MIA rats. 5-HT3A subunit mRNA levels were unchanged.ConclusionThese data suggest descending serotonergic facilitation plays a role in mediating the brush and innocuous mechanical punctate evoked neuronal responses in MIA rats, suggesting an adaptive change in the excitatory serotonergic drive modulating low threshold evoked neuronal responses in MIA-induced OA pain. This alteration in excitatory serotonergic drive, alongside an increase in α2δ-1 mRNA levels, may underlie pregabalins state dependent effects in this model of chronic pain.

Depletion of endogenous spinal 5-HT attenuates the behavioural hypersensitivity to mechanical and cooling stimuli induced by spinal nerve ligation

Abstract There is compelling evidence for a strong facilitatory drive modulating spinal nociceptive transmission. This is in part via serotonergic pathways and originates from the rostroventral medulla. We previously demonstrated that neuropathic pain is associated with an enhanced descending facilitatory drive onto the mechanical evoked responses of dorsal horn neurones, mediated by 5‐HT acting at spinal 5‐HT3 receptors. Furthermore, depletion of spinal 5‐HT has been shown to reduce the at‐level mechanical allodynia that follows spinal cord injury. To further clarify the role and direction of effect of endogenous 5‐HT, we investigated the effects of depleting spinal 5‐HT, via intrathecal injection of 5,7di‐hydroxytryptamine (5,7DHT), on pain behaviours after spinal nerve ligation (SNL). Depletion of spinal 5‐HT in normal animals leads to reductions in mechanical and thermal evoked responses of deep dorsal horn neurones implying that spinal 5‐HT has a predominant facilitatory function. After nerve injury, the frequency of paw withdrawals to low intensity mechanical and cooling stimulation of the ipsilateral hindpaw in the SNL‐5,7DHT group was significantly attenuated when compared with the SNL‐saline group from day seven post‐nerve injury. Sham‐5,7DHT and sham‐saline animals showed very little response sensitivity on either hindpaw. This 5‐HT‐mediated difference in behaviour was independent of both the up‐regulation of the NK1 receptor and spinal microglial activation produced by nerve injury. These data suggest that supraspinal serotonergic influences under these conditions are facilitatory and are implicated in the maintenance of spinal cord neuronal events leading to the behavioural hypersensitivity manifested after peripheral nerve damage.

Local and descending circuits regulate long‐term potentiation and zif268 expression in spinal neurons

Long‐term potentiation (LTP), a use dependent long‐lasting modification of synaptic strength, was first discovered in the hippocampus and later shown to occur in sensory areas of the spinal cord. Here we demonstrate that spinal LTP requires the activation of a subset of superficial spinal dorsal horn neurons expressing the neurokinin‐1 receptor (NK1‐R) that have previously been shown to mediate certain forms of hyperalgesia. These neurons participate in local spinal sensory processing, but are also the origin of a spino‐bulbo‐spinal loop driving a 5‐hydroxytryptamine 3 receptor (5HT3‐R)‐ mediated descending facilitation of spinal pain processing. Using a saporin‐substance P conjugate to produce site‐specific neuronal ablation, we demonstrate that NK1‐R expressing cells in the superficial dorsal horn are crucial for the generation of LTP‐like changes in neuronal excitability in deep dorsal horn neurons and this is modulated by descending 5HT3‐R‐mediated facilitatory controls. Hippocampal LTP is associated with early expression of the immediate‐early gene zif268 and knockout of the gene leads to deficits in long‐term LTP and learning and memory. We found that spinal LTP is also correlated with increased neuronal expression of zif268 in the superficial dorsal horn and that zif268 antisense treatment resulted in deficits in the long‐term maintenance of inflammatory hyperalgesia. Our results support the suggestion that the generation of LTP in dorsal horn neurons following peripheral injury may be one mechanism whereby acute pain can be transformed into a long‐term pain state.

Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis

Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA.

Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Previously reported data have demonstrated that the catalytically active LHN endopeptidase fragment of botulinum neurotoxin type A (termed LHN/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters. Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons. Chemical conjugates prepared between Erythrina cristagalli lectin and LHN/A are assessed in vitro and in in vivo models of pain. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LHN/A, or recombinant LHN/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated. The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin. Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies. Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed. These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics.

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.