Rory Mitchell

Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain.

BACKGROUND Chronic established pain, especially that following nerve injury, is difficult to treat and represents a largely unmet therapeutic need. New insights are urgently required, and we reasoned that endogenous processes such as cooling-induced analgesia may point the way to novel strategies for intervention. Molecular receptors for cooling have been identified in sensory nerves, and we demonstrate here how activation of one of these, TRPM8, produces profound, mechanistically novel analgesia in chronic pain states. RESULTS We show that activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of specific pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, thereby inhibiting the characteristic sensitization of dorsal-horn neurons and behavioral-reflex facilitation. TRPM8 expression was increased in a subset of sensory neurons after nerve injury. The essential role of TRPM8 in suppression of sensitized pain responses was corroborated by specific knockdown of its expression after intrathecal application of an antisense oligonucleotide. We further show that the analgesic effect of TRPM8 activation is centrally mediated and relies on Group II/III metabotropic glutamate receptors (mGluRs), but not opioid receptors. We propose a scheme in which Group II/III mGluRs would respond to glutamate released from TRPM8-containing afferents to exert an inhibitory gate control over nociceptive inputs. CONCLUSIONS TRPM8 and its central downstream mediators, as elements of endogenous-cooling-induced analgesia, represent a novel analgesic axis that can be exploited in chronic sensitized pain states.

An α2 receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurones

Extracellular recordings were made of 59 neurones with long, ascending projections (spinocervical tract (SCT) and dorsal column postsynaptic (DCPS) neurones) in the lumbar dorsal horn of anaesthetized and paralyzed cats. All showed prominent excitatory responses to innocuous stimuli, applied to their cutaneous receptive fields on the ipsilateral hindlimb. The majority of the population investigated (83%) was multireceptive, being activated by noxious as well as innocuous cutaneous stimuli. Drug effects were examined on a regular cycle of responses to these cutaneous stimuli and also to DL-homocysteic acid (DLH). In 49 multireceptive SCT and DCPS neurones, ionophoretically-applied L-noradrenaline (NA) produced a potent selective inhibition of the nociceptive responses (to heat or pinch) in 40 out of 44 SCT and 3 out of 5 DCPS neurones, with no statistically significant change in the responses to innocuous brush or DLH, or in spontaneous activity. NA had no effect on the majority of cells (8 out of 11) that responded only to innocuous stimuli. In 19 SCT neurones that showed NA-selectivity, the alpha 2-selective agonists clonidine (in 12 out of 15) and metaraminol (in 2 out of 3) mimicked this selective effect, whereas, the alpha 1 agonist, phenylephrine and the beta agonist, isoprenaline did not. Furthermore, the alpha 2 antagonists, yohimbine and idazoxan (RX781094), either reversed or reduced the potency of the NA-elicited inhibition of nociceptive responses in all 7 SCT neurones tested. These results are discussed in relation to other evidence for spinal antinociceptive effects of noradrenergic systems acting at a spinal level and the possible involvement of an alpha 2 receptor in such effects.

Rhodopsin-family receptors associate with small G proteins to activate phospholipase D

G-protein-coupled receptors of the rhodopsin family transduce many important neural and endocrine signals. These receptors activate heterotrimeric G proteins and in many cases also cause activation of phospholipase D, an enzyme that can be controlled by the small G proteins ARF and RhoA. Here we show that the activation of phospholipase D that is induced by many, but not all, Ca2+-mobilizing G-protein-coupled receptors is sensitive to inhibitors of ARF and of RhoA. Receptors of this type were co-immunoprecipitated with ARF or RhoA on exposure to agonists, and the effects of GTP analogues on ligand binding to the receptor changed to a profile that is characteristic of small G proteins. These receptors contain the amino-acid sequence AsnProXXTyr in their seventh transmembrane domain, whereas receptors capable of activating phospholipase D without involving ARF contain the sequence AspProXXTyr. Mutation of this latter sequence to AsnProXXTyr in the gonadotropin-releasing hormone receptor conferred sensitivity to an inhibitor of ARF, and the reciprocal mutation in the 5-HT2A receptor for 5-hydroxytryptamine reduced its sensitivity to the inhibitor. Receptors carrying the AsnProXXTyr motif thus seem to form functional complexes with ARF and RhoA.

Antinociceptive actions of descending dopaminergic tracts on cat and rat dorsal horn somatosensory neurones.

1. The actions of dopamine (DA) and DA receptor specific agonists and antagonist ionophoretically applied in the spinal dorsal horn, and of focal electrical stimulation in the region of the supraspinal DA cell groups (A9 and A11) were assessed on the somatosensory responses of dorsal horn neurones, in both the rat and cat. The neurones tested were multireceptive, giving reproducible responses to both noxious (using a mechanical pinch or radiant heat) and innocuous (using a motorized brush) cutaneous stimuli, as well as to ionophoretically applied DL‐homocysteic acid (DLH, a direct excitant). In the cat, all neurones tested were identified as belonging to the spinocervical tract (SCT) and were located in the dorsal horn laminae III‐V, whilst in the rat, spinothalamic tract (STT) and spinomesencephalic (SMT) neurones located in the region of lamina I and laminae III‐V were tested. 2. Ionophoretically applied DA and RU24213, a D2 DA receptor agonist, caused a selective inhibition of the responses to noxious stimuli of SCT, STT and SMT neurones, whilst the responses to non‐nociceptive stimuli, spontaneous activity and DLH‐evoked activity were unaffected. This action was reversed in the presence of sulpiride, the highly selective D2 DA receptor antagonist. Neither sulpiride alone nor SKF38393, a D1 DA receptor agonist, altered evoked or spontaneous activity when ionophoretically applied. 3. Focal electrical stimulation in the region of the A11, but not the A9, DA cell group selectively suppressed nociceptive responses of spinal, multireceptive neurones in the rat. This stimulus‐evoked effect was consistently and rapidly reversed by ionophoresis of sulpiride, in the vicinity of the dorsal horn neurone being tested. In contrast, naloxone and idazoxan (RX781094), an alpha 2‐antagonist, were not effective. 4. This study presents data supporting a selective antinociceptive role for DA at the spinal level, where it has a widespread antinociceptive influence, on cells in both the superficial and deeper dorsal horn. The A11 DA cell group was shown to be a supraspinal site from which a selective antinociceptive action could be electrically evoked and which was mediated by DA at the level of the dorsal horn.

Evidence for a role of metabotropic glutamate receptors in sustained nociceptive inputs to rat dorsal horn neurons.

Several antagonists at metabotropic glutamate (mGlu) receptors, when applied ionophoretically, inhibited the excitation of single dorsal horn neurons elicited by cutaneous administration of the C fibre-selective algogen, mustard oil. The selectivity and stereospecificity of AP3 isomers at mGlu, compared to NMDA receptors was confirmed on responses to agonists and matched by their effects on mustard oil-evoked activity.

The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat

As well as substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have recently been found in the superficial dorsal horn of the spinal cord; NKA originating mainly in fine primary afferents. We have investigated the effects of these tachykinins and a range of analogues on somatosensory responses of single identified dorsal horn neurons, when applied ionophoretically to the region of the substantia gelatinosa. Behavioural reflex tests of thermal nociception were carried out in parallel. The role of NK-1, NK-2 and NK-3 receptors was addressed. NK-1-selective agonists attenuated the non-nociceptive responses of identified multireceptive spinocervical tract (SCT) neurons. Of the endogenous tachykinins, both SP and NKB (a weak NK-1 agonist) showed this effect. No role for NK-3 receptors was identified in our experiments. NK-2-selective agonists (including NKA) caused a unique and selective facilitation of thermal nociceptive responses. NKA also reduced reflex response latency in tail-flick and hot plate tests. NKA as a primary afferent transmitter may thus be involved in mediating or facilitating the expression of thermal nociceptive inputs in the substantia gelatinosa. NKA and SP could be considered as acting in concert in the superficial dorsal horn in an effectively pro-nociceptive modulatory role. Evidence from receptor-selective antagonists supports that obtained with agonists for the roles of particular NK receptors in somatosensory processing. NK-2, but not NK-1 or NK-3 antagonists attenuated endogenous thermal nociceptive responses, supporting the hypothesis that an NK-2 agonist (such as NKA) may normally participate in expression of thermal nociception in the superficial dorsal horn. Behavioural experiments showing increased response latencies with a putative NK-2 selective antagonist further supported the involvement of NK-2 receptors in thermal nociception.

Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

The involvement of metabotropic glutamate receptors and their intracellular signalling pathways in sustained nociceptive transmission in rat dorsal horn neurons

The excitatory responses of individual dorsal horn neurons to cutaneous brush, repeated application of the C-fibre-selective chemical algogen, mustard oil, or to ionophoretic (1S,3R)-ACPD [a metabotropic glutamate receptor (mGluR) agonist] were monitored by extracellular recording. We have previously shown that the responses of dorsal horn neurons to mustard oil are inhibited by several selective antagonists of mGluRs. Effects of ionophoresis of the mGluR antagonists (R,S)-CHPG and L-AP3 and a range of selective inhibitors of intracellular signalling pathways were examined on evoked responses here. The results suggest that protein kinase C, phospholipase A2 and perhaps Ca2+/calmodulin kinase II play a role in mediating the sustained elevated activity of dorsal horn neurons that is incrementally elicited by repeated application of mustard oil, but probably make little contribution to sustained brush-evoked activity. Concurrence in the sensitivity of mustard oil- and (1S,3R)-ACPD-evoked activity to (R,S)-CHPG, L-AP3 and to inhibitors of intracellular signalling pathways, suggests that mGluRs are an important origin of these intracellular signals required for sustained nociception.

Association between raised inflammatory markers and cognitive decline in elderly people with type 2 diabetes: the Edinburgh Type 2 Diabetes Study

OBJECTIVE To determine whether circulating levels of the inflammatory markers C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α are associated with cognitive ability and estimated lifetime cognitive decline in an elderly population with type 2 diabetes. RESEARCH DESIGN AND METHODS A cross-sectional study of 1,066 men and women aged 60–75 years with type 2 diabetes and living in Lothian, Scotland (the Edinburgh Type 2 Diabetes Study), was performed. Seven cognitive tests were used to measure abilities in memory, nonverbal reasoning, information processing speed, executive function, and mental flexibility. The results were used to derive a general intelligence factor (g). A vocabulary–based test was administered as an estimate of peak prior cognitive ability. Results on the cognitive tests were assessed for statistical association with inflammatory markers measured in a venous blood sample at the time of cognitive testing. RESULTS Higher IL-6 and TNF-α levels were associated with poorer age- and sex-adjusted scores on the majority of the individual cognitive tests. They were also associated with g using standardized regression coefficients −0.074 to −0.173 (P < 0.05). After adjusting for vocabulary, education level, cardiovascular dysfunction, duration of diabetes, and glycemic control, IL-6 remained associated with three of the cognitive tests and with g. CONCLUSIONS In this representative population of people with type 2 diabetes, elevated circulating levels of inflammatory markers were associated with poorer cognitive ability. IL-6 levels were also associated with estimated lifetime cognitive decline.

Heterogenous effects of serotonin in the dorsal horn of rat: the involvement of 5-HT1 receptor subtypes

The effect of ionophoretically applied serotonin (5-HT) was tested on cutaneous sensory responses of multireceptive dorsal horn neurones in the anaesthetized rat. Three types of 5-HT action were discerned: selective inhibition of nociceptive responses (10/18 cells), non-selective inhibition of responses to both noxious and innocuous stimuli as well as to excitatory amino acids (4/18 cells) and non-selective excitation of evoked responses (1/18 cells). A few cells (3/18) were unaffected by 5-HT. The use of agonists, shown to discriminate between subtypes of 5-HT1 receptor revealed that a 5-HT1A receptor agonist mimicked the non-selective effects of 5-HT, whereas a 5-HT1B receptor agonist mimicked the selective antinociceptive effects of 5-HT. A 5-HT2 receptor agonist, in contrast, was without effect. Both the selective and the non-selective effects were reversed by a 5-HT1 receptor antagonist, but not a 5-HT2 antagonist.