Nicolas Beaudet

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.

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.

Elucidation of the Structure–Activity Relationships of Apelin: Influence of Unnatural Amino Acids on Binding, Signaling, and Plasma Stability

Apelin is the endogenous ligand of the APJ receptor, a member of the G‐protein‐coupled receptor family. The apelin–APJ complex has been detected in many tissues and is emerging as a promising target for several pathophysiological conditions. There is currently little information on the structure–activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogues of apelin‐13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C‐terminal portion. Analogues were then tested in binding and functional assays by evaluating Gi/o‐mediated decreases in cAMP levels and by assessing β‐arrestin2 recruitment to the APJ receptor. The plasma stability of new compounds was also assessed. Several analogues were found to possess increased binding and higher stability than the parent peptide.

Weight bearing evaluation in inflammatory, neuropathic and cancer chronic pain in freely moving rats

Preclinical pain assessment remains a key step for the development of new and potent painkillers. Significant progress in pain evaluation has been achieved with the development of non-reflexive tools. Seeking efficient and clinically relevant devices for pain-related quality of life assessment, we evaluated a new Dynamic Weight Bearing (DWB) device based on pressure captors in three different preclinical chronic pain models. Inflammatory (CFA), neuropathic (CCI) and bone cancer pain (femoral tumor) models were evaluated in Sprague Dawley rats for mechanical allodynia using dynamic von Frey for pain-related behaviors and DWB for discomfort. We observed similar impairment patterns in all of the models for both von Frey (allodynia) and DWB (weight balance) during the complete observation period, starting at day 3 in CCI- and CFA-affected limbs and at day 14 in bone cancer-afflicted rats, indicating that the DWB could be a useful tool for supporting pain assessment. Interestingly, we demonstrated that the main compensation, when animals experienced pain, was seen in the forepaws, ranging from 46% to 69% of increased load compared to normal. Other pain-related coping behaviors were also measured, such as the time spent on each paw and the contact surface. Our results revealed that CFA, CCI and cancerous rats decreased the use of their ipsilateral hind paws by 30% and showed a 50% reduction in paw surface pressed against the floor. In conclusion, this new device improves methods for preclinical evaluation of discomfort and quality of life proxies and could be helpful in screening putative analgesics.

Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.

Involvement of NTS2 receptors in stress-induced analgesia

Stress activates multiple neural systems that suppress pain sensation. This adaptive phenomenon referred as stress-induced analgesia (SIA) is mediated by the activation of endogenous pain inhibitory systems. Both opioid and non-opioid forms of SIA have been elicited in rodents according to stressor parameters and duration. There is accumulating evidence that the endogenous neurotensin (NT) system plays an important role in SIA. Especially, NT-deficient mice were shown to exhibit reduced SIA following water avoidance or restraint stress. Since central NT produces naloxone-insensitive analgesic effects by acting on spinal and supraspinal NTS2 receptors, we hypothesized that NT might mediate non-opioid SIA through NTS2 activation. Here, we evaluated the influence of an opioid-independent severe stress produced by a cold-water swim for 3 min at 15 degrees C on rodent offsprings pain perception. Our results demonstrated that mice lacking NTS2 exhibit significantly reduced SIA following cold-water swim stress. Indeed, NTS2 knockout mice submitted to both acute (plantar test) and tonic (formalin test) pain stimuli show a greater sensitivity to pain in comparison to wild-type littermates. Accordingly, pretreatment with the NT receptor antagonist SR142948A results in a hyperalgesic response to stress induced by cold-water swim. Endogenous NT regulates hypothalamic-pituitary-adrenal axis activity in stress condition by increasing corticosterone plasma levels. Accordingly, the plasma levels of corticosterone measured by radioimmunoassay are significantly reduced in non-stressed and stressed NTS2-deficient mice in comparison with wild-type mice. To further investigate the site of action of NT in mediating SIA, we microinjected NTS2 agonists in lumbar spinal cord and quantified post-stress sensitivity to pain in rats using the plantar test. Exogenously administered NTS2 analogs, JMV-431, beta-lactotensin and NT69L markedly enhance the magnitude and duration of stress antinociception in both 25- and 60-day-old rats. In sum, by using genetic and pharmacological approaches, we demonstrated here that NTS2 receptors mediate non-opioid SIA. Our results also revealed that the release of endogenous NT in response to stress requires the presence of NTS2 to stimulate corticotropin-releasing factor (CRF)-induced elevation of plasma corticosterone, and that NTS2 receptors localized at the lumbar spinal cord participate to the disinhibition of descending pain control pathways. Therefore, these data highlight the significance of NTS2 as a novel target for the treatment of pain and stress-related disorders.

Behavioral, Medical Imaging and Histopathological Features of a New Rat Model of Bone Cancer Pain

Pre-clinical bone cancer pain models mimicking the human condition are required to respond to clinical realities. Breast or prostate cancer patients coping with bone metastases experience intractable pain, which affects their quality of life. Advanced monitoring is thus required to clarify bone cancer pain mechanisms and refine treatments. In our model of rat femoral mammary carcinoma MRMT-1 cell implantation, pain onset and tumor growth were monitored for 21 days. The surgical procedure performed without arthrotomy allowed recording of incidental pain in free-moving rats. Along with the gradual development of mechanical allodynia and hyperalgesia, behavioral signs of ambulatory pain were detected at day 14 by using a dynamic weight-bearing apparatus. Osteopenia was revealed from day 14 concomitantly with disorganization of the trabecular architecture (µCT). Bone metastases were visualized as early as day 8 by MRI (T1-Gd-DTPA) before pain detection. PET (Na18F) co-registration revealed intra-osseous activity, as determined by anatomical superimposition over MRI in accordance with osteoclastic hyperactivity (TRAP staining). Pain and bone destruction were aggravated with time. Bone remodeling was accompanied by c-Fos (spinal) and ATF3 (DRG) neuronal activation, sustained by astrocyte (GFAP) and microglia (Iba1) reactivity in lumbar spinal cord. Our animal model demonstrates the importance of simultaneously recording pain and tumor progression and will allow us to better characterize therapeutic strategies in the future.