Defining pain and painful sentience in animals

Sentience is essential to most definitions of pain, including a detailed definition invoked by Sneddon et al. to argue that adult and perhaps larval fish feel pain. Because proving painful sentience in non-human animals is not feasible, multiple lines of indirect evidence are needed to implicate pain. This commentary examines the list of 17 criteria used by Sneddon et al. to conclude that fish have conscious pain. The criteria include tests of nociceptive, motivational, and cognitive properties useful for revealing pain-like states that can be understood biologically and be related evolutionarily to human pain. However, additional research is needed to define the crucial aversive component of pain-like states in fish

Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes

Squid have nociceptors that display widespread long-term sensitization and spontaneous activity after bodily injury

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 33 (2013): 10021-10026, doi:10.1523/JNEUROSCI.0646-13.2013.Bodily injury in mammals often produces persistent pain that is driven at least in part by long-lasting sensitization and spontaneous activity (SA) in peripheral branches of primary nociceptors near sites of injury. While nociceptors have been described in lower vertebrates and invertebrates, outside of mammals there is limited evidence for peripheral sensitization of primary afferent neurons, and there are no reports of persistent SA being induced in primary afferents by noxious stimulation. Cephalopod molluscs are the most neurally and behaviorally complex invertebrates, with brains rivaling those of some vertebrates in size and complexity. This has fostered the opinion that cephalopods may experience pain, leading some governments to include cephalopods under animal welfare laws. It is not known, however, if cephalopods possess nociceptors, or whether their somatic sensory neurons exhibit nociceptive sensitization. We demonstrate that squid possess nociceptors that selectively encode noxious mechanical but not heat stimuli, and that show long-lasting peripheral sensitization to mechanical stimuli after minor injury to the body. As in mammals, injury in squid can cause persistent SA in peripheral afferents. Unlike mammals, the afferent sensitization and SA are almost as prominent on the contralateral side of the body as they are near an injury. Thus, while squid exhibit peripheral alterations in afferent neurons similar to those that drive persistent pain in mammals, robust changes far from sites of injury in squid suggest that persistently enhanced afferent activity provides much less information about the location of an injury in cephalopods than it does in mammals.This work was supported by NSF Grants IOS-1146987 to E.T.W. and IOS-1145478 to R.T.H., and the Baxter Pharmaceuticals Fellowship and Bang Summer Research Fellowship from the Marine Biological Laboratory to R.J.C.2013-12-1

Zinc inhibits TRPV1 to alleviate chemotherapy-induced neuropathic pain

Zinc is a transition metal that has a long history of use as an anti-inflammatory agent. It also soothes pain sensations in a number of animal models. However, the effects and mechanisms of zinc on chemotherapy-induced peripheral neuropathy remain unknown. Here we show that locally injected zinc markedly reduces neuropathic pain in male and female mice induced by paclitaxel, a chemotherapy drug, in a TRPV1-dependent manner. Extracellularly applied zinc also inhibits the function of TRPV1 expressed in HEK293 cells and mouse DRG neurons, which requires the presence of zinc-permeable TRPA1 to mediate entry of zinc into the cytoplasm. Moreover, TRPA1 is required for zinc-induced inhibition of TRPV1-mediated acute nociception. Unexpectedly, zinc transporters, but not TRPA1, are required for zinc-induced inhibition of TRPV1-dependent chronic neuropathic pain produced by paclitaxel. Together, our study demonstrates a novel mechanism underlying the analgesic effect of zinc on paclitaxel-induced neuropathic pain that relies on the function of TRPV1

Determinants of TRPV4 Activity following Selective Activation by Small Molecule Agonist GSK1016790A

TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca2+ influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca2+-free medium more significantly at the early stage of the activation rather than the late stage, reflecting apparent partial desensitization. Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca2+-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation

Major Differences in Transcriptional Alterations in Dorsal Root Ganglia Between Spinal Cord Injury and Peripheral Neuropathic Pain Models

Chronic, often intractable, pain is caused by neuropathic conditions such as traumatic peripheral nerve injury (PNI) and spinal cord injury (SCI). These conditions are associated with alterations in gene and protein expression correlated with functional changes in somatosensory neurons having cell bodies in dorsal root ganglia (DRGs). Most studies of DRG transcriptional alterations have utilized PNI models where axotomy-induced changes important for neural regeneration may overshadow changes that drive neuropathic pain. Both PNI and SCI produce DRG neuron hyperexcitability linked to pain, but contusive SCI produces little peripheral axotomy or peripheral nerve inflammation. Thus, comparison of transcriptional signatures of DRGs across PNI and SCI models may highlight pain-associated transcriptional alterations in sensory ganglia that do not depend on peripheral axotomy or associated effects such as peripheral Wallerian degeneration. Data from our rat thoracic SCI experiments were combined with meta-analysis of published whole-DRG RNA-seq datasets from prominent rat PNI models. Striking differences were found between transcriptional responses to PNI and SCI, especially in regeneration-associated genes (RAGs) and long noncoding RNAs (lncRNAs). Many transcriptomic changes after SCI also were found after corresponding sham surgery, indicating they were caused by injury to surrounding tissue, including bone and muscle, rather than to the spinal cord itself. Another unexpected finding was of few transcriptomic similarities between rat neuropathic pain models and the only reported transcriptional analysis of human DRGs linked to neuropathic pain. These findings show that DRGs exhibit complex transcriptional responses to central and peripheral neural injury and associated tissue damage. Although only a few genes in DRG cells exhibited similar changes in expression across all the painful conditions examined here, these genes may represent a core set whose transcription in various DRG cell types is sensitive to significant bodily injury, and which may play a fundamental role in promoting neuropathic pain

Macrophage Migration Inhibitory Factor (MIF) Makes Complex Contributions to Pain-Related Hyperactivity of Nociceptors after Spinal Cord Injury

Neuropathic pain is a major, inadequately treated challenge for people with spinal cord injury (SCI). While SCI pain mechanisms are often assumed to be in the CNS, rodent studies have revealed mechanistic contributions from primary nociceptors. These neurons become chronically hyperexcitable after SCI, generating ongoing electrical activity that promotes ongoing pain. A major question is whether extrinsic chemical signals help to drive ongoing electrical activity after SCI. People living with SCI exhibit acute and chronic elevation of circulating levels of macrophage migration inhibitory factor (MIF), a cytokine implicated in preclinical pain models. Probable nociceptors isolated from male rats and exposed to an MIF concentration reported in human plasma (1 ng/ml) showed hyperactivity similar to that induced by SCI, although, surprisingly, a 10-fold higher concentration failed to increase excitability. Conditioned behavioral aversion to a chamber associated with peripheral MIF injection suggested that MIF stimulates affective pain. A MIF inhibitor, Iso-1, reversed SCI-induced hyperexcitability. Unlike chronic SCI-induced hyperexcitability, acute MIF-induced hyperexcitability was only partially abrogated by inhibiting ERK signaling. Unexpectedly, MIF concentrations that induced hyperactivity in nociceptors from naive animals, after SCI induced a long-lasting conversion from a highly excitable nonaccommodating type to a rapidly accommodating, hypoexcitable type, possibly as a homeostatic response to prolonged depolarization. Treatment with conditioned medium from cultures of DRG cells obtained after SCI was sufficient to induce MIF-dependent hyperactivity in neurons from naive rats. Thus, changes in systemic and DRG levels of MIF may help to maintain SCI-induced nociceptor hyperactivity that persistently promotes pain.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major challenge for people with spinal cord injury (SCI). Pain can drastically impair quality of life, and produces substantial economic and social burdens. Available treatments, including opioids, remain inadequate. This study shows that the cytokine macrophage migration inhibitory factor (MIF) can induce pain-like behavior and plays an important role in driving persistent ongoing electrical activity in injury-detecting sensory neurons (nociceptors) in a rat SCI model. The results indicate that SCI produces an increase in MIF release within sensory ganglia. Low MIF levels potently excite nociceptors, but higher levels trigger a long-lasting hypoexcitable state. These findings suggest that therapeutic targeting of MIF in neuropathic pain states may reduce pain and sensory dysfunction by curbing nociceptor hyperactivity

Reef fishes at all trophic levels respond positively to effective marine protected areas

Marine Protected Areas (MPAs) offer a unique opportunity to test the assumption that fishing pressure affects some trophic groups more than others. Removal of larger predators through fishing is often suggested to have positive flow-on effects for some lower trophic groups, in which case protection from fishing should result in suppression of lower trophic groups as predator populations recover. We tested this by assessing differences in the trophic structure of reef fish communities associated with 79 MPAs and open-access sites worldwide, using a standardised quantitative dataset on reef fish community structure. The biomass of all major trophic groups (higher carnivores, benthic carnivores, planktivores and herbivores) was significantly greater (by 40% - 200%) in effective no-take MPAs relative to fished open-access areas. This effect was most pronounced for individuals in large size classes, but with no size class of any trophic group showing signs of depressed biomass in MPAs, as predicted from higher predator abundance. Thus, greater biomass in effective MPAs implies that exploitation on shallow rocky and coral reefs negatively affects biomass of all fish trophic groups and size classes. These direct effects of fishing on trophic structure appear stronger than any top down effects on lower trophic levels that would be imposed by intact predator populations. We propose that exploitation affects fish assemblages at all trophic levels, and that local ecosystem function is generally modified by fishing

Long-acting injectable Cabotegravir + Rilpivirine for HIV maintenance therapy: Week 48 pooled analysis of phase 3 ATLAS and FLAIR trials

BACKGROUND: Long-acting (LA) injectable regimens are a potential therapeutic option in people living with HIV-1. SETTING: ATLAS (NCT02951052) and FLAIR (NCT02938520) were 2 randomized, open-label, multicenter, multinational phase 3 studies. METHODS: Adult participants with virologic suppression (plasma HIV-1 RNA <50 copies/mL) were randomized (1:1) to continue with their current antiretroviral regimen (CAR) or switch to the long-acting (LA) regimen of cabotegravir (CAB) and rilpivirine (RPV). In the LA arm, participants initially received oral CAB + RPV once-daily for 4 weeks to assess individual safety and tolerability, before starting monthly injectable therapy. The primary endpoint of this combined analysis was antiviral efficacy at week 48 (FDA Snapshot algorithm: noninferiority margin of 4% for HIV-1 RNA ≥50 copies/mL). Safety, tolerability, and confirmed virologic failure (2 consecutive plasma HIV-1 RNA ≥200 copies/mL) were secondary endpoints. RESULTS: The pooled intention-to-treat exposed population included 591 participants in each arm [28% women (sex at birth), 19% aged ≥50 years]. Noninferiority criteria at week 48 were met for the primary (HIV-1 RNA ≥50 copies/mL) and key secondary (HIV-1 RNA <50 copies/mL) efficacy endpoints. Seven individuals in each arm (1.2%) developed confirmed virologic failure; 6/7 (LA) and 3/7 (CAR) had resistance-associated mutations. Most LA recipients (83%) experienced injection site reactions, which decreased in incidence over time. Injection site reactions led to the withdrawal of 6 (1%) participants. The serious adverse event rate was 4% in each arm. CONCLUSION: This combined analysis demonstrates monthly injections of CAB + RPV LA were noninferior to daily oral CAR for maintaining HIV-1 suppression

Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes