Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury.

ABSTRACT: Tissue injuries, including burns, are major causes of death and morbidity worldwide. These injuries result in the release of intracellular molecules and subsequent inflammatory reactions, changing the tissues' chemical milieu and leading to the development of persistent pain through activating pain-sensing primary sensory neurons. However, the majority of pain-inducing agents in injured tissues are unknown. Here, we report that, amongst other important metabolite changes, lysophosphatidylcholines (LPCs) including 18:0 LPC exhibit significant and consistent local burn injury-induced changes in concentration. 18:0 LPC induces immediate pain and the development of hypersensitivities to mechanical and heat stimuli through molecules including the transient receptor potential ion channel, vanilloid subfamily, member 1, and member 2 at least partly via increasing lateral pressure in the membrane. As levels of LPCs including 18:0 LPC increase in other tissue injuries, our data reveal a novel role for these lipids in injury-associated pain. These findings have high potential to improve patient care.

Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin.

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.

Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin.

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.

Ocular Manifestations of Chikungunya Infection: A Systematic Review.

The Chikungunya virus (CHIKV) can cause long lasting symptoms and manifestations. However, there is little information on which ocular ones are most frequent following infection. We performed a systematic review (registered in the International Prospective Register of Systematic Reviews; no CRD42020171928) to establish the most frequent ocular manifestations of CHIKV infection and their associations with gender and age. Articles published until September 2020 were selected from PubMed, Scielo, Cochrane and Scopus databases. Only studies with CHIKV-infected patients and eye alterations were included. Reviews, descriptive studies, or those not investigating the human ocular manifestations of CHIKV, those with patients with other diseases and infections, abstracts and studies without relevant data were excluded. Twenty-five studies were selected for inclusion. Their risk of bias was evaluated by a modified Newcastle-Ottawa scale. The most frequent ocular symptoms of CHIKV infection included ocular pain, inflammation and reduced visual acuity, whilst conjunctivitis and optic neuritis were the most common manifestations of the disease. These occurred mostly in individuals of 42 ± 9.5 years of age and woman. The few available reports on CHIKV-induced eye manifestations highlight the need for further research in the field to gather more substantial evidence linking CHIKV infection, the eye and age/gender. Nonetheless, the data emphasizes that ocular alterations are meaningful occurrences of CHIKV infection which can substantially affect quality of life.

Evidence That a TRPA1-Mediated Murine Model of Temporomandibular Joint Pain Involves NLRP3 Inflammasome Activation.

This study investigates the role of transient receptor potential ankyrin 1 (TRPA1) in murine temporomandibular joint (TMJ) inflammatory hyperalgesia and the influence of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. Two distinct murine models of TMJ pain and inflammation (zymosan and CFA) were established. Spontaneous pain-like behaviours were observed as unilateral front paw cheek wipes. Ipsilateral cheek blood flow was used as a measure of ongoing inflammation, which, to our knowledge, is a novel approach to assessing real-time inflammation in the TMJ. Joint tissue and trigeminal ganglia were collected for ex vivo investigation. Both zymosan and CFA induced a time-dependent increase in hyperalgesia and inflammation biomarkers. Zymosan induced a significant effect after 4 h, correlating with a significantly increased IL-1ß protein expression. CFA (50 µg) induced a more sustained response. The TRPA1 receptor antagonist A967079 significantly inhibited hyper-nociception. The NLRP3 inhibitor MCC950 similarly inhibited hyper-nociception, also attenuating inflammatory markers. In the trigeminal ganglia, CFA-induced CGRP expression showed trends of inhibition by A967079, whilst lba1 immunofluorescence was significantly inhibited by A967079 and MCC950, where the effect of TRPA1 inhibition lasted up to 14 days. Our results show that stimulation of TRPA1 is key to the TMJ pain. However, the inflammasome inhibitor exhibited similar properties in attenuating these pain-like behaviours, in addition to some inflammatory markers. This indicates that in addition to the therapeutic targeting of TRPA1, NLRP3 inhibition may provide a novel therapeutic strategy for TMJ inflammation and pain.

(-)-Englerin-A Has Analgesic and Anti-Inflammatory Effects Independent of TRPC4 and 5.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA's anti-inflammatory and analgesic effects.

Calcitonin Gene-Related Peptide Protects Against Cardiovascular Dysfunction Independently of Nitric Oxide In Vivo.

[Figure: see text].

TRPV1 feed-forward sensitisation depends on COX2 upregulation in primary sensory neurons.

Increased activity and excitability (sensitisation) of a series of molecules including the transient receptor potential ion channel, vanilloid subfamily, member 1 (TRPV1) in pain-sensing (nociceptive) primary sensory neurons are pivotal for developing pathological pain experiences in tissue injuries. TRPV1 sensitisation is induced and maintained by two major mechanisms; post-translational and transcriptional changes in TRPV1 induced by inflammatory mediators produced and accumulated in injured tissues, and TRPV1 activation-induced feed-forward signalling. The latter mechanism includes synthesis of TRPV1 agonists within minutes, and upregulation of various receptors functionally linked to TRPV1 within a few hours, in nociceptive primary sensory neurons. Here, we report that a novel mechanism, which contributes to TRPV1 activation-induced TRPV1-sensitisation within ~ 30 min in at least ~ 30% of TRPV1-expressing cultured murine primary sensory neurons, is mediated through upregulation in cyclooxygenase 2 (COX2) expression and increased synthesis of a series of COX2 products. These findings highlight the importance of feed-forward signalling in sensitisation, and the value of inhibiting COX2 activity to control pain, in nociceptive primary sensory neurons in tissue injuries.

Examining the role of transient receptor potential canonical 5 (TRPC5) in osteoarthritis.

INTRODUCTION: Osteo-arthritis (OA) involves joint degradation and usually pain; with mechanisms poorly understood and few treatment options. There is evidence that the transient receptor potential canonical 5 (TRPC5) mRNA expression is reduced in OA patients' synovia. Here we examine the profile of TRPC5 in DRG and involvement in murine models of OA. DESIGN: TRPC5 KO mice were subjected to partial meniscectomy (PMNX) or injected with monoiodoacetate (MIA) and pain-related behaviours were determined. Knee joint pathological scores were analysed and gene expression changes in ipsilateral synovium and dorsal root ganglia (DRG) determined. c-Fos protein expression in the ipsilateral dorsal horn of the spinal cord was quantified. RESULTS: TRPC5 KO mice developed a discrete enhanced pain-related phenotype. In the MIA model, the pain-related phenotype correlated with c-Fos expression in the dorsal horn and increased expression of nerve injury markers ATF3, CSF1 and galanin in the ipsilateral DRG. There were negligible differences in the joint pathology between WT and TRPC5 KO mice, however detailed gene expression analysis determined increased expression of the mast cell marker CD117 as well as extracellular matrix remodelling proteinases MMP2, MMP13 and ADAMTS4 in MIA-treated TRPC5 KO mice. TRPC5 expression was defined to sensory subpopulations in DRG. CONCLUSIONS: Deletion of TRPC5 receptor signalling is associated with exacerbation of pain-like behaviour in OA which correlates with increased expression of enzymes involved in extracellular remodelling, inflammatory cells in the synovium and increased neuronal activation and injury in DRG. Together, these results identify a modulating role for TRPC5 in OA-induced pain-like behaviours.

The Pharmacology of Pain Associated With the Monoiodoacetate Model of Osteoarthritis.

The high incidence of osteoarthritis (OA) in an increasingly elderly population anticipates a dramatic rise in the number of people suffering from this disease in the near future. Because pain is the main reason patients seek medical help, effective pain management-which is currently lacking-is paramount to improve the quality of life that OA sufferers desperately seek. Good animal models are, in this day and age, fundamental tools for basic research of new therapeutic pathways. Several animal models of OA have been characterized, but none of them reproduces entirely all symptoms of the disease. Choosing between different animal models depends largely on which aspect of OA one aims to study. Here, we review the current understanding of the monoiodoacetate (MIA) model of OA. MIA injection in the knee joint leads to the progressive disruption of cartilage, which, in turn, is associated with the development of pain-like behavior. There are several reasons why the MIA model of OA seems to be the most adequate to study the pharmacological effect of new drugs in pain associated with OA. First, the pathological changes induced by MIA share many common traits with those observed in human OA (Van Der Kraan et al., 1989; Guingamp et al., 1997; Guzman et al., 2003), including loss of cartilage and alterations in the subchondral bone. The model has been extensively utilized in basic research, which means that the time course of pain-related behaviors and histopathological changes, as well as pharmacological profile, namely of commonly used pain-reducing drugs, is now moderately understood. Also, the severity of the progression of pathological changes can be controlled by grading the concentration of MIA administered. Further, in contrast with other OA models, MIA offers a rapid induction of pain-related phenotypes, with the cost-saving consequence in new drug screening. This model, therefore, may be more predictive of clinical efficacy of novel pharmacological tools than other chronic or acute OA models.

A historical perspective on the role of sensory nerves in neurogenic inflammation.

The term 'neurogenic inflammation' is commonly used, especially with respect to the role of sensory nerves within inflammatory disease. However, despite over a century of research, we remain unclear about the role of these nerves in the vascular biology of inflammation, as compared with their interacting role in pain processing and of their potential for therapeutic manipulation. This chapter attempts to discuss the progress in understanding, from the initial discovery of sensory nerves until the present day. This covers pioneering findings that these nerves exist, are involved in vascular events and act as important sensors of environmental changes, including injury and infection. This is followed by discovery of the contents they release such as the established vasoactive neuropeptides substance P and CGRP as well as anti-inflammatory peptides such as the opioids and somatostatin. The more recent emergence of the importance of the transient receptor potential (TRP) channels has revealed some of the mechanisms by which these nerves sense environmental stimuli. This knowledge enables a platform from which to learn of the potential role of neurogenic inflammation in disease and in turn of novel therapeutic targets.

Phosphorylated Histone 3 at Serine 10 Identifies Activated Spinal Neurons and Contributes to the Development of Tissue Injury-Associated Pain.

Transcriptional changes in superficial spinal dorsal horn neurons (SSDHN) are essential in the development and maintenance of prolonged pain. Epigenetic mechanisms including post-translational modifications in histones are pivotal in regulating transcription. Here, we report that phosphorylation of serine 10 (S10) in histone 3 (H3) specifically occurs in a group of rat SSDHN following the activation of nociceptive primary sensory neurons by burn injury, capsaicin application or sustained electrical activation of nociceptive primary sensory nerve fibres. In contrast, brief thermal or mechanical nociceptive stimuli, which fail to induce tissue injury or inflammation, do not produce the same effect. Blocking N-methyl-D-aspartate receptors or activation of extracellular signal-regulated kinases 1 and 2, or blocking or deleting the mitogen- and stress-activated kinases 1 and 2 (MSK1/2), which phosphorylate S10 in H3, inhibit up-regulation in phosphorylated S10 in H3 (p-S10H3) as well as fos transcription, a down-stream effect of p-S10H3. Deleting MSK1/2 also inhibits the development of carrageenan-induced inflammatory heat hyperalgesia in mice. We propose that p-S10H3 is a novel marker for nociceptive processing in SSDHN with high relevance to transcriptional changes and the development of prolonged pain.

Inflammation of peripheral tissues and injury to peripheral nerves induce differing effects in the expression of the calcium-sensitive N-arachydonoylethanolamine-synthesizing enzyme and related molecules in rat primary sensory neurons.

Elevation of intracellular Ca2+ concentration induces the synthesis of N-arachydonoylethanolamine (anandamide) in a subpopulation of primary sensory neurons. N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is the only known enzyme that synthesizes anandamide in a Ca2+ -dependent manner. NAPE-PLD mRNA as well as anandamide's main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1), the inhibitory cannabinoid type 1 (CB1) receptor, and the main anandamide-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), are all expressed by subpopulations of nociceptive primary sensory neurons. Thus, NAPE-PLD, TRPV1, the CB1 receptor, and FAAH could form an autocrine signaling system that could shape the activity of a major subpopulation of nociceptive primary sensory neurons, contributing to the development of pain. Although the expression patterns of TRPV1, the CB1 receptor, and FAAH have been comprehensively elucidated, little is known about NAPE-PLD expression in primary sensory neurons under physiological and pathological conditions. This study shows that NAPE-PLD is expressed by about one-third of primary sensory neurons, the overwhelming majority of which also express nociceptive markers as well as the CB1 receptor, TRPV1, and FAAH. Inflammation of peripheral tissues and injury to peripheral nerves induce differing but concerted changes in the expression pattern of NAPE-PLD, the CB1 receptor, TRPV1, and FAAH. Together these data indicate the existence of the anatomical basis for an autocrine signaling system in a major proportion of nociceptive primary sensory neurons and that alterations in that autocrine signaling by peripheral pathologies could contribute to the development of both inflammatory and neuropathic pain.

Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their Crosstalk.

The cannabinoid type 1 (CB1) receptor and the capsaicin receptor (TRPV1) exhibit co-expression and complex, but largely unknown, functional interactions in a sub-population of primary sensory neurons (PSN). We report that PSN co-expressing CB1 receptor and TRPV1 form two distinct sub-populations based on their pharmacological properties, which could be due to the distribution pattern of the two receptors. Pharmacologically, neurons respond either only to capsaicin (COR neurons) or to both capsaicin and the endogenous TRPV1 and CB1 receptor ligand anandamide (ACR neurons). Blocking or deleting the CB1 receptor only reduces both anandamide- and capsaicin-evoked responses in ACR neurons. Deleting the CB1 receptor also reduces the proportion of ACR neurons without any effect on the overall number of capsaicin-responding cells. Regarding the distribution pattern of the two receptors, neurons express CB1 and TRPV1 receptors either isolated in low densities or in close proximity with medium/high densities. We suggest that spatial distribution of the CB1 receptor and TRPV1 contributes to the complexity of their functional interaction.

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse.

A major symptom of patients with osteoarthritis (OA) is pain that is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain. The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Anandamide in primary sensory neurons: too much of a good thing?

The quest for possible targets for the development of novel analgesics has identified the activation of the cannabinoid type 1 (CB1) receptor outside the CNS as a potential means of providing relief from persistent pain, which currently constitutes an unmet medical need. Increasing tissue levels of the CB1 receptor endogenous ligand N-arachidonoylethanolamine (anandamide), by inhibiting anandamide degradation through blocking the anandamide-hydrolysing enzyme fatty acid amide hydrolase, has been suggested to be used to activate the CB1 receptor. However, recent clinical trials revealed that this approach does not deliver the expected relief from pain. Here, we discuss one of the possible reasons, the activation of the transient receptor potential vanilloid type 1 ion channel (TRPV1) on nociceptive primary sensory neurons (PSNs) by anandamide, which may compromise the beneficial effects of increased tissue levels of anandamide. We conclude that better design such as concomitant blocking of anandamide hydrolysis and anandamide uptake into PSNs, to inhibit TRPV1 activation, could overcome these problems.

Anandamide produced by Ca(2+)-insensitive enzymes induces excitation in primary sensory neurons.

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca(2+)-sensitive and Ca(2+)-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca(2+)-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca(2+). We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca(2+)-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca(2+)-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca(2+)-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.