Livio Luongo

The endovanilloid/endocannabinoid system in human osteoclasts: Possible involvement in bone formation and resorption

Recent studies suggest a role for the endocannabinoid/endovanilloid anandamide in the regulation of bone resorption/formation balance in mice. Here, we examined the co-expression of the transient receptor potential vanilloid type 1 (TRPV1) and the cannabinoid CB1/CB2 receptors together with N-acylphosphatidylethanolamine-hydrolizing phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), the two enzymes responsible of the synthesis and catabolism of anandamide respectively, in human osteoclasts. Co-expression of TRPV1, CB1/CB2, NAPE-PLD and FAAH was found in both human osteoclast cultures and in native osteoclasts from human bone biopsies. Moreover, agonist-evoked calcium entry indicated that the TRPV1 receptor is functionally active in vitro. Consistently, biomolecular and functional experiments showed that resiniferatoxin (RTX), a selective TRPV1 receptor agonist, increased the expression and the activity of TRAP and cathepsin K, two specific osteoclast biomarkers. The evidence that cannabinoid and vanilloid receptors are co-expressed in human osteoclasts suggests that they might cross-talk to modulate the intrinsic balance of bone mineralization and resorption by different actions of anandamide through TRPV1 and cannabinoid receptors. The presence of the endocannabinoid/endovanilloid proteins in human osteoclasts will likely have implications for the management of bone demineralization associated syndrome (i. e. osteoporosis).

TRPV1-Dependent and -Independent Alterations in the Limbic Cortex of Neuropathic Mice: Impact on Glial Caspases and Pain Perception

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

Moving towards supraspinal TRPV1 receptors for chronic pain relief

Transient receptor potential vanilloid type 1 (TRPV1) receptor is a non selective ligand-gated cation channel activated by capsaicin, heat, protons and endogenous lipids termed endovanilloids. As well as peripheral primary afferent neurons and dorsal root ganglia, TRPV1 receptor is also expressed in spinal and supraspinal structures such as those belonging to the endogenous antinociceptive descending pathway which is a circuitry of the supraspinal central nervous system whose task is to counteract pain. It includes periaqueductal grey (PAG) and rostral ventromedial medulla (RVM) whose activation leads to analgesia. Such an effect is associated with a glutamate increase and the activation of OFF and inhibition of ON cell population in the rostral ventromedial medulla (RVM). Activation of the antinociceptive descending pathway via TPRV1 receptor stimulation in the PAG may be a novel strategy for producing analgesia in chronic pain. This review will summarize the more recent insights into the role of TRPV1 receptor within the antinociceptive descending pathway and its possible exploitation as a target for new pain-killer agents in chronic pain conditions, with particular emphasis on the most untreatable pain state: neuropathic pain.

Palmitoylethanolamide reduces formalin-induced neuropathic-like behaviour through spinal glial/microglial phenotypical changes in mice.

Palmitoylethanolamide (PEA) is an endogenous cannabinoid-like compound in the central nervous system, which can modulate several functions in different pathological states, such as inflammation and pain response. We have here investigated the effect of PEA (5-10 mg/kg, intraperitoneally) on mechanical allodynia and thermal hyperalgesia 3 and 7 days following peripheral injection of formalin. Formalin induced a significant decrease of thermal and mechanical threshold in the injected and contralateral paw. PEA chronic treatment (once per day) significantly reduced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. Consistently, in vivo electrophysiological analysis revealed a significant increase of the duration and frequency, and a rapid decrease in the onset of evoked activity of the spinal nociceptive neurons 7 days after formalin. PEA normalized the electrophysiological parameters in a dose-dependent manner. Moreover, we investigated PEA effect on the glial/microglial phenotypical changes associated with spinal neuronal sensitization. We found that formalin induced a significant microglia and glia activation normalized by PEA, together with increased expression of glial interleukin 10. Finally, primary microglial cell cultures, conditioned with PEA or vehicle, where transplanted in naive and formalin-treated mice, and nociceptive neurons were recorded. We observed that only PEA-conditioned cells normalized the activity of sensitized nociceptive neurons. In conclusion these data confirm the potent anti-inflammatory and anti-allodynic effect of PEA, and highlight a possible targeted microglial/glial effect of this drug in the spinal cord.

Palmitoylethanolamide reduces granuloma-induced hyperalgesia by modulation of mast cell activation in rats

The aim of this study was to obtain evidences of a possible analgesic role for palmitoylethanolamide (PEA) in chronic granulomatous inflammation sustained by mast cell (MC) activation in rats at 96 hours. PEA (200-400-800 μg/mL), locally administered at time 0, reduced in a concentration-dependent manner the expression and release of NGF in comparison with saline-treated controls. PEA prevented nerve formation and sprouting, as shown by histological analysis, reduced mechanical allodynia, evaluated by Von Frey filaments, and inhibited dorsal root ganglia activation. These results were supported by the evidence that MCs in granuloma were mainly degranulated and closely localized near nerve fibres and PEA significantly reduced MC degranulation and nerves fibre formation. These findings are the first evidence that PEA, by the modulation of MC activation, controls pain perception in an animal model of chronic inflammation, suggesting its potential use for the treatment of all those painful conditions in which MC activation is an initial key step.

Long-lasting effects of human mesenchymal stem cell systemic administration on pain-like behaviors, cellular, and biomolecular modifications in neuropathic mice.

Background: Neuropathic pain (NP) is an incurable disease caused by a primary lesion in the nervous system. NP is a progressive nervous system disease that results from poorly defined neurophysiological and neurochemical changes. Its treatment is very difficult. Current available therapeutic drugs have a generalized nature, sometime acting only on the temporal pain properties rather than targeting the several mechanisms underlying the generation and propagation of pain. Methods: Using biomolecular and immunohistochemical methods, we investigated the effect of the systemic injection of human mesenchymal stem cells (hMSCs) on NP relief. We used the spared nerve injury (SNI) model of NP in the mouse. hMSCs were injected into the tail vein of the mouse. Stem cell injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored every 10 days starting from day 11 until 90 days after surgery. Results: hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once injected into the tail vein. An anti-nociceptive effect was detectable from day 11 post surgery (7 days post cell injection). hMSCs were mainly able to home in the spinal cord and pre-frontal cortex of neuropathic mice. Injected hMSCs reduced the protein levels of the mouse pro-inflammatory interleukin IL-1β and IL-17 and increased protein levels of the mouse anti-inflammatory interleukin IL-10, and the marker of alternatively activated macrophages CD106 in the spinal cord of SNI mice. Conclusion: As a potential mechanism of action of hMSCs in reducing pain, we suggest that they could exert their beneficial action through a restorative mechanism involving: (i) a cell-to-cell contact activation mechanism, through which spinal cord homed hMSCs are responsible for switching pro-inflammatory macrophages to anti-inflammatory macrophages; (ii) secretion of a broad spectrum of molecules to communicate with other cell types. This study could provide novel findings in MSC pre-clinical biology and their therapeutic potential in regenerative medicine.

Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states

Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.

The A1 adenosine receptor as a new player in microglia physiology

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2X4,7 and P2Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca2+ influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive‐specific neuron before and after application of resting or ATP‐treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone. GLIA 2014;62:122–132

Effects of intra-ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α (PPAR-α) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-α antagonist, GW6471 and by a large-conductance Ca(2+)-activated K(+) channel inhibitor, charybdotoxin. Cannabinoid 1 (CB(1)) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV(1)) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-α receptors mediate, and CB(1) and TRPV(1) receptors antagonise, PEA-induced effects within the PAG-RVM circuitry.

The antinociceptive effect of 2-chloro-2′-C-methyl-N6-cyclopentyladenosine (2′-Me-CCPA), a highly selective adenosine A1 receptor agonist, in the rat

Abstract This study was undertaken in order to investigate the effect of 2‐chloro‐2′‐C‐methyl‐N6‐cyclopentyladenosine (2′‐Me‐CCPA), a potent and highly selective adenosine A1 receptor agonist, on nociceptive responses and on the ongoing or tail flick‐related changes of rostral ventromedial medulla (RVM) ON‐ and OFF‐cell activities. Systemic administrations of 2′‐Me‐CCPA (2.5–5 mg/kg, i.p.) reduced the nociceptive response in the plantar and formalin tests, in a way prevented by DPCPX (3 mg/kg, i.p.), a selective A1 receptor antagonist. Similarly, intra‐periaqueductal grey (PAG) 2′‐Me‐CCPA (0.5–1–2 nmol/rat) reduced pain behaviour in the plantar and formalin tests, in a way inhibited by DPCPX (0.5 nmol/rat). Moreover, when administered systemically (2.5–5 mg/kg, i.p.) or intra‐PAG (0.5–1 nmol/rat) 2′‐Me‐CCPA increased the tail flick latencies, delayed the tail flick‐related onset of the ON‐cell burst and decreased the duration of the OFF‐cell pause in a dose dependent manner. Furthermore, it decreased RVM ON‐cell and increased OFF‐cell ongoing activities. The in vivo electrophysiological effects were all prevented by DPCPX (0.5 nmol/rat). This study confirms the role of adenosine A1 receptors in modulating pain and suggests a critical involvement of these receptors within PAG–RVM descending pathway for the processing of pain.