Philippe Sarret

CCL2 Released from Neuronal Synaptic Vesicles in the Spinal Cord Is a Major Mediator of Local Inflammation and Pain after Peripheral Nerve Injury

CCL2 chemokine and its receptor CCR2 may contribute to neuropathic pain development. We tested the hypothesis that injury to peripheral nerves triggers CCL2 release from afferents in the dorsal horn spinal cord (DHSC), leading to pronociceptive effects, involving the production of proinflammatory factors, in particular. Consistent with the release of CCL2 from primary afferents, electron microscopy showed the CCL2 immunoreactivity in glomerular boutons and secretory vesicles in the DHSC of naive rats. Through the ex vivo superfusion of DHSC slices, we demonstrated that the rate of CCL2 secretion was much lower in neonatal capsaicin-treated rats than in controls. Thus, much of the CCL2 released in the DHSC originates from nociceptive fibers bearing TRPV1 (transient receptor potential vanilloid 1). In contrast, high levels of CCL2 released from the DHSC were observed in neuropathic pain animal model induced by chronic constriction of the sciatic nerve (SN-CCI). The upregulated expression of proinflammatory markers and extracellular signal-regulated kinase (ERK) 1/2 pathway activation (ERK1/2 phosphorylation) in the DHSC of SN-CCI animals were reversed by intrathecal administration of the CCR2 antagonist INCB3344 (N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide). These pathological pain-associated changes in the DHSC were mimicked by the intrathecal injection of exogenous CCL2 in naive rats and were prevented by the administration of INCB3344 or ERK inhibitor (PD98059). Finally, mechanical allodynia, which was fully developed 2 weeks after SN-CCI in rats, was attenuated by the intrathecal injection of INCB3344. Our data demonstrate that CCL2 has the typical characteristics of a neuronal mediator involved in nociceptive signal processing and that antagonists of its receptor are promising agents from treating neuropathic pain.

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP‐1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP‐1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP‐1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co‐localized with pain‐related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium‐dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2‐induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.

Neurochemokines: a menage a trois providing new insights on the functions of chemokines in the central nervous system

J. Neurochem. (2011) 118, 680–694.

DEVELOPMENTAL MOTOR DEFICITS INDUCED BY COMBINED FETAL EXPOSURE TO LIPOPOLYSACCHARIDE AND EARLY NEONATAL HYPOXIA/ISCHEMIA : A NOVEL ANIMAL MODEL FOR CEREBRAL PALSY IN VERY PREMATURE INFANTS

A critical issue in animal models of perinatal brain injury is to adapt the pertinent pathophysiological scenarios to their corresponding developmental window in order to induce neuropathological and behavioral characteristics reminiscent to perinatal cerebral palsy (CP). A major problem in most of these animal models designed up to now is that they do not present motor deficits characteristic of CP. Using a unique rat paradigm of prenatal inflammation combined to an early postnatal hypoxia-ischemia pertinent to the context of very early premature human newborns, we were interested in finding out if such experimental conditions might reproduce both histological damages and behavioral deficits previously described in the human context. We showed that exposure to lipopolysaccharide (LPS) or hypoxia-ischemia (H/I) induced behavioral alterations in animals subjected to forced motor activity. When both LPS and H/I aggressions were combined, the motor deficits reached their highest intensity and affected both spontaneous and forced motor activities. LPS+H/I-exposed animals also showed extensive bilateral cortical and subcortical lesions of the motor networks affecting the frontal cortices and underlying white matters fascicles, lenticular nuclei and the substantia nigra. These neuropathological lesions and their associated motor behavioral deficits are reminiscent of those observed in very preterm human neonates affected by subsequent CP and validate the value of the present animal model to test new therapeutic strategies which might open horizons for perinatal neuroprotection.

Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines

The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT‐PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa‐type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.

The Chemokine CCL2 Increases Nav1.8 Sodium Channel Activity in Primary Sensory Neurons through a Gβγ-Dependent Mechanism

Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Nav1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Nav1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Nav1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Nav1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gβγ inhibitor), indicating the involvement of Gβγ released from PTX-sensitive Gi/o-proteins in the cross talk between CCR2 and Nav1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Nav1.8 currents via a CCR2/Gβγ-dependent mechanism.

Increased anxiety-like behaviors in rats experiencing chronic inflammatory pain.

For many patients, chronic pain is often accompanied, and sometimes amplified, by co-morbidities such as anxiety and depression. Although it represents important challenges, the establishment of appropriate preclinical behavioral models contributes to drug development for treating chronic inflammatory pain and associated psychopathologies. In this study, we investigated whether rats experiencing persistent inflammatory pain induced by intraplantar injection of complete Freunds adjuvant (CFA) developed anxiety-like behaviors, and whether clinically used analgesic and anxiolytic drugs were able to reverse CFA-induced anxiety-related phenotypes. These behaviors were evaluated over 28 days in both CFA- and saline-treated groups with a variety of behavioral tests. CFA-induced mechanical allodynia resulted in increased anxiety-like behaviors as evidenced by: (1) a significant decrease in percentage of time spent and number of entries in open arms of the elevated-plus maze (EPM), (2) a decrease in number of central squares visited in the open field (OF), and (3) a reduction in active social interactions in the social interaction test (SI). The number of entries in closed arms in the EPM and the distance traveled in the OF used as indicators of locomotor performance did not differ between treatments. Our results also reveal that in CFA-treated rats, acute administration of morphine (3mg/kg, s.c.) abolished tactile allodynia and anxiety-like behaviors, whereas acute administration of diazepam (1mg/kg, s.c) solely reversed anxiety-like behaviors. Therefore, pharmacological treatment of anxiety-like behaviors induced by chronic inflammatory pain can be objectively evaluated using multiple behavioral tests. Such a model could help identify/validate alternative potential targets that influence pain and cognitive dimensions of anxiety.

Chemokine Network in the Nervous System: A New Target for Pain Relief

Physical insults including but not limited to nerve damage, inflammation, visceral pathologies and cancer generate long lasting pain commonly referred as chronic pain. Recently, members of the chemokine family and their receptors emerged as key modulators in nociceptive influx transmission in neuropathic and inflammatory chronic pain models. To this day, rodents defective in specific chemokine receptors have provided evidence of the implication of chemokine in pain sensitivity. In addition, up-regulation of chemokines and their receptors at multiple levels in the central nervous (CNS) and peripheral (PNS) systems is associated in the development of chronic pain. Indeed, we point out the fact that chemokines are synthesized and released by both neuronal and non-neuronal cells and act as neuromodulators. Even if their functional roles in the CNS remain largely unknown, chemokines participate in the glial activation and modulation of neuronal excitability as well as neurotransmitter release. This review focuses on three chemokines (i.e. CCL2, CXCL12, CX3CL1) recently identified as important mediators of the initiation and maintenance of pain hypersensitivity, thus broadening the panel of new strategies for the management of chronic pain.

Postnatal administration of IL-1Ra exerts neuroprotective effects following perinatal inflammation and/or hypoxic-ischemic injuries

New therapeutic strategies are needed to protect neonates, especially premature newborns, against brain injury and associated neurobehavioral deficits. The role of pro-inflammatory cytokines, especially IL-1β, in the pathophysiological pathway leading to neonatal brain damage is increasingly recognized and represents an attractive therapeutic target. We investigated the therapeutic potential of postnatal systemic administration of the interleukin (IL)-1 receptor antagonist (IL-1Ra) in an animal model of perinatal brain injury using the insults most common to human neonates, i.e. prenatal exposure to inflammation and/or postnatal hypoxia-ischaemia (HI). We found that postnatal administration of IL-1Ra preserved motor function and exploratory behavior after either prenatal exposure to inflammatory agent lipopolysaccharide (LPS) or postnatal HI insult. The deleterious effect of combined prenatal LPS and postnatal HI on brain development was also alleviated by administration of IL-1Ra, as seen by the protected neural stem cell population, prevention of myelin loss in the internal capsule, decreased gliosis, and decreased neurobehavioral impairment. This study showed the distinct pattern of functional deficits induced by prenatal inflammation as compared to postnatal HI and the therapeutic potential of IL-1Ra administration against neonatal brain injury. Furthermore, our results highlight the potential for postnatal treatment of prenatal inflammatory stressors.

Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors.

Intrathecal administration of the neuropeptide neurotensin (NT) was shown previously to exert antinociceptive effects in a variety of acute spinal pain paradigms including hotplate, tail-flick, and writhing tests. In the present study, we sought to determine whether some of these antinociceptive effects might be elicited via stimulation of low-affinity NTS2 receptors. We first established, using immunoblotting and immunohistochemical techniques, that NTS2 receptors were extensively associated with putative spinal nociceptive pathways, both at the level of the dorsal root ganglia and of the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal administration of NT or selective NTS2 agonists on acute thermal pain. Both NT and NTS2 agonists, levocabastine and Boc-Arg-Arg-Pro-Tyrψ(CH2NH)Ile-Leu-OH (JMV-431), induced dose-dependent antinociceptive responses in the tail-flick test. The effects of levocabastine and of JMV-431 were unaffected by coadministration of the NTS1-specific antagonist 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy-phenyl)pyrazol-3-yl)carboxylamino]tricyclo)3.3.1.1.3.7)-decan-2-carboxylic acid (SR48692), confirming that they were NTS2 mediated. In contrast, the antinociceptive effects of NT were partly abolished by coadministration of SR48692, indicating that NTS1 and NTS2 receptors were both involved. These results suggest that NTS2 receptors play a role in the regulation of spinal nociceptive inputs and that selective NTS2 agonists may offer new avenues for the treatment of acute pain.