Transgenic mouse behavioural analysis has furthered our understanding of the molecular and cellular mechanisms underlying damage sensing and pain. However, it is not unusual for conflicting data on the pain phenotypes of knockout mice to be generated by reputable groups. Here we focus on some technical aspects of measuring mouse pain behaviour that are often overlooked, which may help explain discrepancies in the pain literature. We examined touch perception using von Frey hairs and mechanical pain thresholds using the Randall-Selitto test. Thermal pain thresholds were measured using the Hargreaves apparatus and a thermal place preference test. Sodium channel Nav1.7 knockout mice show a mechanical deficit in the hairy skin, but not the paw, whilst shaving the abdominal hair abolished this phenotype. Nav1.7, Nav1.8 and Nav1.9 knockout mice show deficits in noxious mechanosensation in the tail, but not the paw. TRPA1 knockout mice, however, have a loss of noxious mechanosensation in the paw but not the tail. Studies of heat and cold sensitivity also show variability depending on the intensity of the stimulus. Deleting Nav1.7, Nav1.8 or Nav1.9 in Nav1.8-positive sensory neurons attenuates responses to slow noxious heat ramps, whilst responses to fast noxious heat ramps are only reduced when Nav1.7 is lost in large diameter sensory neurons. Deleting Nav1.7 from all sensory neurons attenuates responses to noxious cooling but not extreme cold. Finally, circadian rhythms dramatically influence behavioural outcome measures such as von Frey responses, which change by 80% over the day. These observations demonstrate that fully characterising the phenotype of a transgenic mouse strain requires a range of behavioural pain models. Failure to conduct behavioural tests at different anatomical locations, stimulus intensities, and at different points in the circadian cycle may lead to a pain behavioural phenotype being misinterpreted, or missed altogether.

Multiple studies support the pro-nociceptive role of brain-derived neurotrophin factor (BDNF) in pain processes in the peripheral and central nervous system. We have previously shown that nociceptor-derived BDNF is implicated in inflammatory pain. Microglial-derived BDNF has also been shown to be involved in neuropathic pain. However, the distinct contribution of primary afferent-derived BNDF to chronic pain processing remains undetermined. In this study, we used Advillin-CreERT2 mice to delete Bdnf from all adult peripheral sensory neurons. Conditional BDNF knockouts were healthy with no sensory neuron loss. Behavioural assays and in vivo electrophysiology indicated that spinal excitability was normal. Following formalin inflammation or neuropathy with a modified Chung model, we observed normal development of acute pain behaviour, but a deficit in second phase formalin-induced nocifensive responses and a reversal of neuropathy-induced mechanical hypersensitivity during the later chronic pain phase in conditional BDNF knockout mice. In contrast, we observed normal development of acute and chronic neuropathic pain in the Seltzer model, indicating differences in the contribution of BDNF to distinct models of neuropathy. We further used a model of hyperalgesic priming to examine the contribution of primary afferent-derived BDNF in the transition from acute to chronic pain, and found that primed BDNF knockout mice do not develop prolonged mechanical hypersensitivity. Our data suggest that BDNF derived from sensory neurons plays a critical role in mediating the transition from acute to chronic pain.

Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs.

Human genetic studies have implicated the voltage-gated sodium channel Na V 1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na V 1.7 (IC 50 0.9 nM) with at least 40–1000-fold selectivity over all other Na V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na V 1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na V 1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na V 1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na V 1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

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.

Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.

Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.

Human acute and inflammatory pain requires the expression of voltage-gated sodium channel Nav1.7 but its significance for neuropathic pain is unknown. Here we show that Nav1.7 expression in different sets of mouse sensory and sympathetic neurons underlies distinct types of pain sensation. Ablating Nav1.7 gene (SCN9A) expression in all sensory neurons using Advillin-Cre abolishes mechanical pain, inflammatory pain and reflex withdrawal responses to heat. In contrast, heat-evoked pain is retained when SCN9A is deleted only in Nav1.8-positive nociceptors. Surprisingly, responses to the hotplate test, as well as neuropathic pain, are unaffected when SCN9A is deleted in all sensory neurons. However, deleting SCN9A in both sensory and sympathetic neurons abolishes these pain sensations and recapitulates the pain-free phenotype seen in humans with SCN9A loss-of-function mutations. These observations demonstrate an important role for Nav1.7 in sympathetic neurons in neuropathic pain, and provide possible insights into the mechanisms that underlie gain-of-function Nav1.7-dependent pain conditions.

The activity of voltage-gated sodium channels has long been linked to disorders of neuronal excitability such as epilepsy and chronic pain. Recent genetic studies have now expanded the role of sodium channels in health and disease, to include autism, migraine, multiple sclerosis, cancer as well as muscle and immune system disorders. Transgenic mouse models have proved useful in understanding the physiological role of individual sodium channels, and there has been significant progress in the development of subtype selective inhibitors of sodium channels. This review will outline the functions and roles of specific sodium channels in electrical signalling and disease, focusing on neurological aspects. We also discuss recent advances in the development of selective sodium channel inhibitors.

The molecular mechanisms determining magnitude and duration of inflammatory pain are still unclear. We assessed the contribution of G protein-coupled receptor kinase (GRK)-6 to inflammatory hyperalgesia in mice. We showed that GRK6 is a critical regulator of severity and duration of cytokine-induced hyperalgesia. In GRK6−/− mice, a significantly lower dose (100 times lower) of intraplantar interleukin (IL)-1β was sufficient to induce hyperalgesia compared with wild-type (WT) mice. In addition, IL-1β hyperalgesia lasted much longer in GRK6−/− mice than in WT mice (8 d in GRK6−/− versus 6 h in WT mice). Tumor necrosis factor (TNF)-α-induced hyperalgesia was also enhanced and prolonged in GRK6−/− mice. In vitro, IL-1β-induced p38 phosphorylation in GRK6−/− dorsal root ganglion (DRG) neurons was increased compared with WT neurons. In contrast, IL-1β only induced activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway in WT neurons, but not in GRK6−/− neurons. In vivo, p38 inhibition attenuated IL-1β- and TNF-α-induced hyperalgesia in both genotypes. Notably, however, whereas PI 3-kinase inhibition enhanced and prolonged hyperalgesia in WT mice, it did not have any effect in GRK6-deficient mice. The capacity of GRK6 to regulate pain responses was also apparent in carrageenan-induced hyperalgesia, since thermal and mechanical hypersensitivity was significantly prolonged in GRK6−/− mice. Finally, GRK6 expression was reduced in DRGs of mice with chronic neuropathic or inflammatory pain. Collectively, these findings underline the potential role of GRK6 in pathological pain. We propose the novel concept that GRK6 acts as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation.

Background: Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase. Results: We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents. Conclusions: Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.

Pain afflicts a fifth of the population, and animal models have proven useful in target validation and analgesic drug development. Thresholds to pain are tested by applying a sensory stimulus, such as heat or pressure, and observing the resulting withdrawal behavior. Sensitized pain models involve provoking an inflammatory response or damaging the nerves themselves, and testing the changes in pain threshold. In this article, mouse models of acute mechanical and thermal pain and inflammatory, visceral, and neuropathic pain are discussed. These behavioral measures can be used to phenotype transgenic mice for target validation and mechanistic studies, as well as to screen potential analgesic compounds. Curr. Protoc. Mouse Biol. 1:383-412 © 2011 by John Wiley & Sons, Inc.