Resurgent neuropathic discharge: an obstacle to the therapeutic use of neuroma resection?

ABSTRACT: Ectopic discharge ("ectopia") in damaged afferent axons is a major contributor to chronic neuropathic pain. Clinical opinion discourages surgical resection of nerves proximal to the original injury site for fear of resurgence of ectopia and exacerbated pain. We tested this concept in a well-established animal neuroma model. Teased-fiber recordings were made of ectopic spontaneous discharge originating in the experimental nerve-end neuroma and associated dorsal root ganglia in rats that underwent either a single transection (with ligation) of the sciatic nerve or 2 consecutive transections separated by 7, 14, 21, or 30 days. Ectopia emerged in afferent A and C fibers after a single cut with kinetics anticipated from previous studies. When resection was performed during the early period of intense A-fiber activity, a brief period of resurgence was observed. However, resection of neuromas of more than 14 days was followed by low levels of activity with no indication of resurgence. This remained the case in trials out to 60 days after the first cut. Similarly, we saw no indication of resurgent ectopia originating in axotomized dorsal root ganglion neuronal somata and no behavioral reflection of resurgence. In summary, we failed to validate the concern that proximal resection of a problematic nerve would lead to intense resurgent ectopic discharge and pain. As the well-entrenched concept of resurgence is based more on case reports and anecdotes than on solid evidence, it may be justified to relax the stricture against resecting neuromas as a therapeutic strategy, at least within the framework of controlled clinical trials.

Anesthetic loss of consciousness induced by chemogenetic excitation of mesopontine effector neurons.

Although general anesthesia is normally induced by systemic dosing, an anesthetic state can be induced in rodents by microinjecting minute quantities of GABAergic agents into the brainstem mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesions to the MPTA render rats relatively insensitive to standard anesthetic doses delivered systemically. Using a chemogenetic approach we have identified and characterized a small subpopulation of neurons restricted to the MPTA which, when excited, render the animal anesthetic by sensorimotor (immobility) and electroencephalographic (EEG) criteria. These "effector-neurons" do not express GABAAδ-Rs, the likely target of GABAergic anesthetics. Rather, we report a distinct sub-population of nearby MPTA neurons which do. During anesthetic induction these likely excite the effector-neurons by disinhibition. Within the effector population ~ 70% appear to be glutamatergic, ~30% GABAergic and ~ 40% glycinergic. Most are projection neurons that send ascending or descending axons to distant targets associated with the individual functional components of general anesthesia: atonia, analgesia, amnesia, and loss-of-consciousness.

A nodal point for brain-state transitions: the mesopontine tegmental anesthesia area (MPTA) in mice.

The mesopontine tegmental anesthesia area (MPTA) was identified in rats as a singular brainstem locus at which microinjection of minute quantities of GABAergic agents rapidly and reversibly induces loss-of-consciousness and a state of general anesthesia, while lesioning renders animals insensitive to anesthetics at normal systemic doses. Obtaining similar results in mice has been challenging, however, slowing research progress on how anesthetics trigger brain-state transitions. We have identified roadblocks that impeded translation from rat to mouse and tentatively located the MPTA equivalent in this second species. We describe here a series of modifications to the rat protocol that allowed us to document pro-anesthetic changes in mice following localized stereotactic delivery of minute quantities (20 nL) of the GABAA-receptor agonist muscimol into the brainstem mesopontine tegmentum. The optimal locus identified proved to be homologous to the MPTA in rats, and local neuronal populations in rats and mice were similar in size and shape. This outcome should facilitate application of the many innovative gene-based methodologies available primarily in mice to the study of how activity in brainstem MPTA neurons brings about anesthetic loss-of-consciousness and permits pain-free surgery.

Paradoxical anesthesia: Sleep-like EEG during anesthesia induced by mesopontine microinjection of GABAergic agents.

General anesthetic agents are thought to induce loss-of-consciousness (LOC) and enable pain-free surgery by acting on the endogenous brain circuitry responsible for sleep-wake cycling. In clinical use, the entire CNS is exposed to anesthetic molecules with LOC and amnesia usually attributed to synaptic suppression in the cerebral cortex and immobility and analgesia to agent action in the spinal cord and brainstem. This model of patch-wise suppression has been challenged, however, by the observation that all functional components of anesthesia can be induced by focal delivery of minute quantities of GABAergic agonists to the brainstem mesopontine tegmental anesthesia area (MPTA). We compared spectral features of the cortical electroencephalogram (EEG) in rats during systemic anesthesia and anesthesia induced by MPTA microinjection. Systemic administration of (GABAergic) pentobarbital yielded the sustained, δ-band dominant EEG signature familiar in clinical anesthesia. In contrast, anesthesia induced by MPTA microinjection (pentobarbital or muscimol) featured epochs of δ-band EEG alternating with the wake-like EEG, the pattern typical of natural non-rapid-eye-movement (NREM) and REM sleep. The rats were not sleeping, however, as they remained immobile, atonic and unresponsive to noxious pinch. Recalling the paradoxical wake-like quality the EEG during REM sleep, we refer to this state as "paradoxical anesthesia". GABAergic anesthetics appear to co-opt both cortical and spinal components of the sleep network via dedicated axonal pathways driven by MPTA neurons. Direct drug exposure of cortical and spinal neurons is not necessary, and is probably responsible for off-target side-effects of systemic administration including monotonous δ-band EEG, hypothermia and respiratory depression. SIGNIFICANCE STATEMENT: The concept that GABAergic general anesthetic agents induce loss-of-consciousness by substituting for an endogenous neurotransmitter, thereby co-opting neural circuitry responsible for sleep-wake transitions, has gained considerable traction. However, the electroencephalographic (EEG) signatures of sleep and anesthesia differ fundamentally. We show that when the anesthetic state is generated by focal delivery of GABAergics into the mesopontine tegmental anesthesia area (MPTA) the resulting EEG repeatedly transitions between delta-wave-dominant and wake-like patterns much as in REM-NREM sleep. This suggests that systemic (clinical) anesthetic delivery, which indiscriminately floods the entire cerebrum with powerful inhibitory agents, obscures the sleep-like EEG signature associated with the less adulterated form of anesthesia obtained when the drugs are applied selectively to loci where the effective neurotransmitter substitution actually occurs.

Individual Mesopontine Neurons Implicated in Anesthetic Loss-of-consciousness Employ Separate Ascending Pathways to the Cerebral Cortex.

The mesopontine tegmental anesthesia area (MPTA) is a small brainstem nucleus that, when exposed to minute quantities of GABAA receptor agonists, induces a state of general anesthesia. In addition to immobility and analgesia this state is accompanied by widespread suppression of neural activity in the cerebral cortex and high delta-band power in the electroencephalogram. Collectively, MPTA neurons are known to project to a variety of forebrain targets which are known to relay to the cortex in a highly distributed manner. Here we ask whether ascending projections of individual MPTA neurons collateralize to several of these cortical relay nuclei, or access only one. Using rats, contrasting retrograde tracers were microinjected pairwise on one side into three ascending relays: the basal forebrain, the zona incerta-lateral hypothalamus and the intralaminar thalamic nuclear group. In addition, in separate animals, each target was microinjected bilaterally. MPTA neurons were then identified as being single-or double-labeled, indicating projection to one target nucleus or collateralization to both. Results indicated that double-labeling was rare, occurring on average in only 1.3% of the neurons sampled. The overwhelming majority of individual MPTA neurons showed specific connectivity, contributing to only one of the major ascending pathways, either ipsilaterally or contralaterally, but not bilaterally. This architecture would permit particular functional aspects of anesthetic loss-of-consciousness to be driven by specific subpopulations of MPTA neurons.

Reduced Sensitivity to Anesthetic Agents upon Lesioning the Mesopontine Tegmental Anesthesia Area in Rats Depends on Anesthetic Type.

BACKGROUND: The brainstem mesopontine tegmental anesthesia area is a key node in circuitry responsible for anesthetic induction and maintenance. Microinjecting the γ-aminobutyric acid-mediated (GABAergic) anesthetic pentobarbital in this nucleus rapidly and reversibly induces general anesthesia, whereas lesioning it renders the animal relatively insensitive to pentobarbital administered systemically. This study investigated whether effects of lesioning the mesopontine tegmental anesthesia area generalize to other anesthetic agents. METHODS: Cell-selective lesions were made using ibotenic acid, and rats were later tested for changes in the dose-response relation to etomidate, propofol, alfaxalone/alfadolone, ketamine, and medetomidine delivered intravenously using a programmable infusion pump. Anesthetic induction for each agent was tracked using five behavioral endpoints: loss of righting reflex, criterion for anesthesia (score of 11 or higher), criterion for surgical anesthesia (score of 14 or higher), antinociception (loss of pinch response), and deep surgical anesthesia (score of 16). RESULTS: As reported previously for pentobarbital, on-target mesopontine tegmental anesthesia area lesions reduced sensitivity to the GABAergic anesthetics etomidate and propofol. The dose to achieve a score of 16 increased to 147 ± 50% of baseline in control animals ± SD (P = 0.0007; 7 lesioned rats and 18 controls) and 136 ± 58% of baseline (P = 0.010; 6 lesioned rats and 21 controls), respectively. In contrast, responsiveness to the neurosteroids alfaxalone and alfadolone remained unchanged compared with baseline (94 ± 24%; P = 0.519; 6 lesioned rats and 18 controls) and with ketamine increased slightly (90 ± 11%; P = 0.039; 6 lesioned rats and 19 controls). The non-GABAergic anesthetic medetomidine did not induce criterion anesthesia even at the maximal dose tested. The dose to reach the maximal anesthesia score actually obtained was unaffected by the lesion (112 ± 8%; P = 0.063; 5 lesioned rats and 18 controls). CONCLUSIONS: Inability to induce anesthesia in lesioned animals using normally effective doses of etomidate, propofol, and pentobarbital suggests that the mesopontine tegmental anesthesia area is the effective target of these, but not necessarily all, GABAergic anesthetics upon systemic administration. Cortical and spinal functions are likely suppressed by recruitment of dedicated ascending and descending pathways rather than by direct, distributed drug action.

Enhanced wakefulness following lesions of a mesopontine locus essential for the induction of general anesthesia.

The induction of general anesthesia shares many features with the transition from wakefulness to non-rapid eye movement (NREM) sleep, suggesting that the two types of brain-state transition are orchestrated by a common neuronal mechanism. Previous studies revealed a brainstem locus, the mesopontine tegmental anesthesia area (MPTA), that is of singular importance for anesthetic induction. Microinjection of GABAergic anesthetics there induces rapid loss-of-consciousness and lesions render the animal relatively insensitive to anesthetics administered systemically. Here we show that MPTA lesions also alter the natural sleep-wake rhythm by increasing overall wake time at the expense of time asleep (NREM and REM sleep equally), with nearly all of the change occurring during the dark hours of the light-dark cycle. The effect was proportional to the extent of the lesion and was not seen after lesions just outside of the MPTA, or following sham lesions. Thus, MPTA neurons appear to play a role in natural bistable brain-state switching (sleep-wake) as well as in loss and recovery of consciousness induced pharmacologically.

Mesopontine Neurons Implicated in Anesthetic Loss-of-consciousness have Either Ascending or Descending Axonal Projections, but Not Both.

The MPTA (mesopontine tegmental anesthesia area) is a key node in a network of axonal pathways that collectively engage the key components of general anesthesia: immobility and atonia, analgesia, amnesia and loss-of-consciousness. In this study we have applied double retrograde tracing to analyze MPTA connectivity, with a focus on axon collateralization. Prior tracer studies have shown that collectively, MPTA neurons send descending projections to spinal and medullary brain targets associated with atonia and analgesia as well as ascending projections to forebrain structures associated with amnesia and arousal. Here we ask whether individual MPTA neurons collateralize broadly as might be expected of modulatory circuitry, sending axonal branches to both caudal and to rostral targets, or whether connectivity is more selective. Two distinguishable retrograde tracers were microinjected into pairs ("dyads") of known synaptic targets of the MPTA, one caudal and one rostral. We found that neurons that were double-labeled, and hence project to both targets were rare, constituting <0.5% on average of all MPTA neurons that project to these targets. The large majority sent axons either caudally, presumably to mediate mobility and/or antinociception, or rostrally, presumably to mediate mnemonic and/or arousal/cognitive functions. MPTA neurons with descending vs ascending projections also differed in size and shape, supporting the conclusion that they constitute distinct neuronal populations. From these and prior observations we conclude that the MPTA has a hybrid architecture including neurons with heterogeneous patterns of connectivity, some highly collateralized and some more targeted.

Location of the Mesopontine Neurons Responsible for Maintenance of Anesthetic Loss of Consciousness.

The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinociception). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats by microinjection of small amounts of GABAA-receptor agonists into an upper brainstem region named the mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here, using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state transitions.SIGNIFICANCE STATEMENT General anesthesia permits pain-free surgery. Furthermore, because anesthetic agents have the unique ability to reversibly switch the brain from wakefulness to a state of unconsciousness, knowing how and where they work is a potential route to unraveling the neural mechanisms that underlie awareness itself. Using a novel method, we have located a small, and apparently one of a kind, cluster of neurons in the mesopontine tegmentum that are capable of effecting brain-state switching when exposed to GABAA-receptor agonists. This action appears to be mediated by a network of dedicated axonal pathways that project directly and/or indirectly to nearby arousal nuclei of the brainstem and to more distant targets in the forebrain and spinal cord.

Mesopontine Switch for the Induction of General Anesthesia by Dedicated Neural Pathways.

We review evidence that the induction of anesthesia with GABAergic agents is mediated by a network of dedicated axonal pathways, which convey a suppressive signal to remote parts of the central nervous system. The putative signal originates in an anesthetic-sensitive locus in the brainstem that we refer to as the mesopontine tegmental anesthesia area (MPTA). This architecture stands in contrast to the classical notion that anesthetic molecules themselves directly mediate anesthetic induction after global distribution by the vascular circulation. The MPTA came to light in a systematic survey of the rat brain as a singular locus at which microinjection of minute quantities of GABAergic anesthetics is able to reversibly induce a state resembling surgical anesthesia. The rapid onset of anesthesia, the observed target specificity, and the fact that effective doses are far too small to survive dilution during vascular redistribution to distant areas in the central nervous system are all incompatible with the classical global suppression model. Lesioning the MPTA selectively reduces the animal's sensitivity to systemically administered anesthetics. Taken together, the microinjection data show that it is sufficient to deliver γ-aminobutyric acid A receptor (GABAA-R) agonists to the MPTA to induce an anesthesia-like state and the lesion data indicate that MPTA neurons are necessary for anesthetic induction by the systemic route at clinically relevant doses. Known connectivity of the MPTA provides a scaffold for defining the specific projection pathways that mediate each of the functional components of anesthesia. Because MPTA lesions do not induce coma, the MPTA is not a key arousal nucleus essential for maintaining the awake state. Rather, it appears be a "gatekeeper" of arousal function, a major element in a flip-flop switching mechanism that executes rapid and reversible transitions between the awake and the anesthetic state.

Transient loss of consciousness during hypercapnia and hypoxia: Involvement of pathways associated with general anesthesia.

Transient loss of consciousness (TLOC), frequently triggered by perturbation in essential physiological parameters such as pCO2 or O2, is considered a passive consequence of generalized degradation in high-level cerebral functioning. However, the fact that it is almost always accompanied by atonia and loss of spinal nocifensive reflexes suggests that it might actually be part of a "syndrome" mediated by neural circuitry, and ultimately be adaptive. Widespread suppression by molecules distributed in the vasculature is also the classical explanation of general anesthesia. Recent data, however, suggest that anesthesia is due, rather, to drug action at a specific brainstem locus, the mesopontine tegmental anesthesia area (MPTA), with the spectrum of anesthetic effects resulting from secondary recruitment of specific axonal pathways. If so, might the MPTA also be involved in TLOC induced by hypercapnia and hypoxia? We exposed rats to gas mixtures that provoke hypercapnia and hypoxia and asked whether cell-selective lesions of the MPTA affect TLOC. Entry into TLOC, monitored as time to loss of the righting reflex (LORR) was unaffected. However, resumption of the righting reflex (RORR), and of response to pinch stimuli (ROPR), was significantly delayed. The extent of both effects correlated with the extent of damage in the MPTA, but was unrelated to damage that extended beyond the borders of the MPTA. The results implicate neurons in a specific common-core region of the MPTA in TLOC induced by both forms of asphyxia. This is the same area responsible for general anesthesia induced by GABAergic anesthetic agents. This implies the involvement of a common set of brain nuclei and dedicated axonal pathways, rather than nonspecific global suppression, in the mechanism mediating all three instances of TLOC.

Model of anaesthetic induction by unilateral intracerebral microinjection of GABAergic agonists.

General anaesthetic agents induce loss of consciousness coupled with suppression of movement, analgesia and amnesia. Although these diverse functions are mediated by neural structures located in wide-ranging parts of the neuraxis, anaesthesia can be induced rapidly and reversibly by bilateral microinjection of minute quantities of γ-aminobutyric acid (GABA)A -R agonists at a small, focal locus in the mesopontine tegmentum (MPTA). State switching under these circumstances is presumably executed by dedicated neural pathways and does not require widespread distribution of the anaesthetic agent itself, the classical assumption regarding anaesthetic induction. Here it was asked whether these pathways serve each hemisphere independently, or whether there is bilateral redundancy such that the MPTA on each side is capable of anaesthetizing the entire brain. Either of two GABAA -R ligands were microinjected unilaterally into the MPTA in awake rats, the barbiturate modulator pentobarbital and the direct receptor agonist muscimol. Both agents, microinjected on either side, induced clinical anaesthesia, including bilateral atonia, bilateral analgesia and bilateral changes in cortical activity. The latter was monitored using c-fos expression and electroencephalography. This action, however, was not simply a consequence of suppressing spike activity in MPTA neurons, as unilateral (or bilateral) microinjection of the local anaesthetic lidocaine at the same locus failed to induce anaesthesia. A model of the state-switching circuitry that accounts for the bilateral action of unilateral microinjection and also for the observation that inactivation with lidocaine is not equivalent to inhibition with GABAA -R agonists was proposed. This is a step in defining the overall switching circuitry that underlies anaesthesia.

Brainstem node for loss of consciousness due to GABA(A) receptor-active anesthetics.

The molecular agents that induce loss of consciousness during anesthesia are classically believed to act by binding to cognate transmembrane receptors widely distributed in the CNS and critically suppressing local processing and network connectivity. However, previous work has shown that microinjection of anesthetics into a localized region of the brainstem mesopontine tegmentum (MPTA) rapidly and reversibly induces anesthesia in the absence of global spread. This implies that functional extinction is determined by neural pathways rather than vascular distribution of the anesthetic agent. But does clinical (systemic-induced) anesthesia employ MPTA-linked circuitry? Here we show that cell-selective lesioning of the MPTA in rats does not, in itself, induce anesthesia or coma. However, it increases the systemic dose of pentobarbital required to induce anesthesia, in a manner proportional to the extent of the lesion. Such lesions also affect emergence, extending the duration of anesthesia. Off-target and sham lesions were ineffective. Combined with the prior microinjection data, we conclude that drug delivery to the MPTA is sufficient to induce loss-of-consciousness and that neurons in this locus are necessary for anesthetic induction at clinically relevant doses. Together, the results support an architecture for anesthesia with the MPTA serving as a key node in an endogenous network of dedicated pathways that switch between wake and unconsciousness. As such, the MPTA might also play a role in syncope, concussion and sleep.

Dynamic genotype-selective "phenotypic switching" of CGRP expression contributes to differential neuropathic pain phenotype.

Using a genetic model we demonstrate the role played by "phenotypic switching" of calcitonin gene related peptide (CGRP) expression in axotomized large Aß afferents in the development of neuropathic pain behavior in rats. After nerve injury both substance P and CGRP are upregulated in Aß afferents in the corresponding DRGs. It has been proposed that intraspinal release of these neurotransmitters upon gentle stroking of skin drives ascending pain signaling pathways resulting in tactile allodynia. We reported previously that in rat lines genetically selected for high (HA) vs. low (LA) pain phenotype, SP is upregulated equally in both strains, but that CGRP is upregulated exclusively in the pain prone HA line (Nitzan-Luques et al., 2011). This implicates CGRP as the principal driver of tactile allodynia. Here we confirm this conclusion by showing: 1) that the time of emergence of CGRP-IR in DRG Aß neurons and their central terminals in HA rats matches that of pain behavior, 2) that following spinal nerve lesion (SNL) selective activation of low threshold afferents indeed drives postsynaptic pain-signaling neurons and induces central sensitization in HA rats, as monitored using c-Fos as a marker. These changes are much less prominent in LA rats, 3) that intrathecal (i.t.) administration of CGRP induces tactile allodynia in naïve rats and 4) that i.t. administration of the CGRP-receptor antagonist BIBN4096BS (Olcegepant) attenuates SNL-evoked tactile allodynia, without blocking baseline nociception. Together, these observations support the hypothesis that genotype-selective phenotypic switching of CGRP expression in Aß afferents following nerve injury is a fundamental mechanism of neuropathic tactile allodynia.

Spontaneous pain in partial nerve injury models of neuropathy and the role of nociceptive sensory cover.

Spontaneous pain is difficult to measure in animals. One proposed biomarker of spontaneous pain is autotomy, a behavior frequently observed in rats with complete hindpaw denervation (the neuroma model of neuropathic pain). A large body of evidence suggests that this behavior reflects spontaneous dysesthesic sensations akin to phantom limb pain or anesthesia dolorosa. After partial paw denervation, such as in the spared nerve injury (SNI) model of neuropathic pain, autotomy is rare. Does this mean that spontaneous pain is absent? We denervated hindpaws in two stages: SNI surgery completed 7 or 28 days later by transection of the saphenous and sural nerves (SaSu). Minimal autotomy was evoked by the first stage. But it started rapidly after SaSu surgery rendered the limb numb, much more rapidly than after denervation in a single stage (neuroma model). The acceleration was proportional to the delay between the two surgeries. This "priming" effect of the first surgery indicates that the neural substrate of autotomy, spontaneous neuropathic pain, was not initiated by the onset of numbness, but rather by the first, SNI surgery. But the animal's pain experience was occult. The saphenous and sural nerves provided nociceptive sensory cover for the paw, preventing the behavioral expression of the spontaneous pain in the form of autotomy. The results support prior observations suggesting that partial nerve injury triggers spontaneous pain as well as allodynia, and illustrate the importance of nociceptive sensory cover in the prevention of self-inflicted limb injury.

Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2.

Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca(2+) channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.

Evaluation of the antiallodynic, teratogenic and pharmacokinetic profile of stereoisomers of valnoctamide, an amide derivative of a chiral isomer of valproic acid.

The purpose of this study was to evaluate the stereoselective pain relieving (antiallodynic) activity, antiallodynic-anticonvulsant correlation, teratogenicity and pharmacokinetic profile of two stereoisomers of valnoctamide (VCD), a CNS-active amide derivative of a chiral isomer of valproic acid (VPA). The individual stereoisomers (diastereomers), (2R,3S)-VCD and (2S,3S)-VCD were synthesized and their antiallodynic activity was evaluated in rats using the spinal nerve ligation model of neuropathic pain. The pharmacokinetic profile of the two stereoisomers was evaluated in rats following: 1) i.p. administration of racemic-VCD, 2) i.p. administration of the individual stereoisomers (2R,3S)-VCD and (2S,3S)-VCD. Teratogenicity of racemic-VCD and its two individual stereoisomers was evaluated in a SWV mouse strain known to be highly susceptible to VPA-induced teratogenicity. Racemic-VCD, (2R,3S)-VCD and (2S,3S)-VCD showed a dose-related reversal of tactile allodynia with ED(50) values of 52, 61 and 39 mg/kg, respectively. (2S,3S)-VCD was significantly more potent than (2R,3S)-VCD but the opposite is true for its anticonvulsant-effect. In the teratogenicity evaluation racemic-VCD and its two individual stereoisomers showed mild embryotoxicity at doses 7-10 times higher than their antiallodynic-ED(50) values, while (2S,3S)-VCD was significantly less embryotoxic than (2R,3S)-VCD and racemic-VCD. Following administration of the racemic-VCD there was an increase in the primary pharmacokinetic parameters of (2S,3S)-VCD but not of (2R,3S)-VCD. This study demonstrates that both racemic-VCD and its stereoisomers show high potency as antiallodynic compounds and possess a wide safety margin. (2S,3S)-VCD is more potent and less embryotoxic than (2R,3S)-VCD and thus, has a potential to become a candidate for development as a new drug for treating neuropathic pain.

Evaluation of the enantioselective antiallodynic and pharmacokinetic profile of propylisopropylacetamide, a chiral isomer of valproic acid amide.

Propylisopropylacetamide (PID) is a chiral CNS-active constitutional isomer of valpromide, the amide derivative of the major antiepileptic drug valproic acid (VPA). The purpose of this work was: a) To evaluate enantiospecific activity of PID on tactile allodynia in the Chung (spinal nerve ligation, SNL) model of neuropathic pain in rats; b) To evaluate possible sedation at effective antiallodynic doses, using the rotorod ataxia test; c) To investigate enantioselectivity in the pharmacokinetics of (R)- and (S)-PID in comparison to (R,S)-PID; and d) To determine electrophysiologically whether PID has the potential to affect tactile allodynia by suppressing ectopic afferent discharge in the peripheral nervous system (PNS). (R)-, (S)- and (R,S)-PID produced dose-related reversal of tactile allodynia with ED(50) values of 46, 48, 42 mg/kg, respectively. The individual PID enantiomers were not enantioselective in their antiallodynic activity. No sedative side-effects were observed at these doses. Following i.p. administration of the individual enantiomers, (S)-PID had lower clearance (CL) and volume of distribution (V) and a shorter half-life (t(1/2)) than (R)-PID. However following administration of (R,S)-PID, both enantiomers had similar CL and V, but (R)-PID had a longer t(1/2). Systemic administration of (R,S)-PID at antiallodynic doses did not suppress spontaneous ectopic afferent discharge generated in the injured peripheral nerve, suggesting that its antiallodynic action is exerted in the CNS rather than the PNS. Both of PID's enantiomers, and the racemate, are more potent antiallodynic agents than VPA and have similar potency to gabapentin. Consequently, they have the potential to become new drugs for treating neuropathic pain.

pain2: A neuropathic pain QTL identified on rat chromosome 2.

We aimed to locate a chronic pain-associated QTL in the rat (Rattus norvegicus) based on previous findings of a QTL (pain1) on chromosome 15 of the mouse (Mus musculus). The work was based on rat selection lines HA (high autotomy) and LA (low autotomy) which show a contrasting pain phenotype in response to nerve injury in the neuroma model of neuropathic pain. An F(2) segregating population was generated from HA and LA animals. Phenotyped F(2) rats were genotyped on chromosome 7 and chromosome 2, regions that share a partial homology with mouse chromosome 15. Our interval mapping analysis revealed a LOD score value of 3.63 (corresponding to p=0.005 after correcting for multiple testing using permutations) on rat chromosome 2, which is suggestive of the presence of a QTL affecting the predisposition to neuropathic pain. This QTL was mapped to the 14-26cM interval of chromosome 2. Interestingly, this region is syntenic to mouse chromosome 13, rather than to the region of mouse chromosome 15 that contains pain1. This chromosomal position indicates that it is possibly a new QTL, and hence we name it pain2. Further work is needed to replicate and to uncover the underlying gene(s) in both species.

Sex-specific variability and a 'cage effect' independently mask a neuropathic pain quantitative trait locus detected in a whole genome scan.

Sex and environment may dramatically affect genetic studies, and thus should be carefully considered. Beginning with two inbred mouse strains with contrasting phenotype in the neuroma model of neuropathic pain (autotomy), we established a backcross population on which we conducted a genome-wide scan. The backcross population was partially maintained in small social groups and partially in isolation. The genome scan detected one previously reported quantitative trait locus (QTL) on chromosome 15 (pain1), but no additional QTLs were found. Interestingly, group caging introduced phenotypic noise large enough to completely mask the genetic effect of the chromosome 15 QTL. The reason appears to be that group-caging animals from the low-autotomy strain together with animals from the high-autotomy strain dramatically increases autotomy in the otherwise low-autotomy mice (males or females). The converse, suppression of pain behaviour in the high-autotomy strain when caged with the low-autotomy strain was also observed, but only in females. Even in isolated mice, the genetic effect of the chromosome 15 QTL was significant only in females. To determine why, we evaluated autotomy levels of females in 12 different inbred stains of mice and compared them to previously reported levels for males. Strikingly larger environmental variation was observed in males than in females for this pain phenotype. The high baseline variance in males can explain the difficulty in detecting the genetic effect, which was readily seen in females. Our study emphasizes the importance of sex and environment in the genetic analysis of pain.