Karine Thibault

Characterisation of sensory abnormalities observed in an animal model of multiple sclerosis: a behavioural and pharmacological study.

Multiple sclerosis is a chronic inflammatory demyelinating disease, associated, in 50–80% of patients, with persistent pain. While the type of pain that affects these patients is being more documented, the mechanisms underlying this pathology are still poorly understood and animal models of such chronic pain associated with MS are required. The aim of our study was to characterize the sensory abnormalities and in particular the clinical signs linked to persistent pain in two models of Experimental Autoimmune Encephalomyelitis (EAE) in the rat. This behavioural characterization tested several sensory modalities such as mechanical and thermal (heat/cold) hyperalgesia or allodynia and explored some of these modalities on two different extremities: the hindpaws and the tail. Our study showed that while one of the model produced more robust motor impairment, animals of both models suffer from mechanical hyperalgesia and thermal allodynia to cold, both at the level of the tail and the hindpaws. While the time‐course changes of some of these modalities are shifted in the time between the two models, they represent good models of the sensory abnormalities experienced by MS patients. The second part of our study aimed at characterizing from a pharmacological point of view the most robust model (“EAE+Cyclosporine”) and showed that Gabapentin, Duloxetine and Tramadol partially relieved some of the clinical signs. Our results suggest that the model “EAE+Cyclosporine” in the rat is a good model of chronic sensory abnormalities observed in MS patients both from a behavioural and pharmacological point of view.

BDNF-dependent plasticity induced by peripheral inflammation in the primary sensory and the cingulate cortex triggers cold allodynia and reveals a major role for endogenous BDNF as a tuner of the affective aspect of pain.

Painful experiences are multilayered, composed of sensory, affective, cognitive and behavioral facets. Whereas it is well accepted that the development of chronic pain is due to maladaptive neuronal changes, the underlying molecular mechanisms, their relationship to the different pain modalities, and indeed the localization of these changes are still unknown. Brain-derived neurotrophic factor (BDNF) is an activity-dependent neuromodulator in the adult brain, which enhances neuronal excitability. In the spinal cord, BDNF underlies the development and maintenance of inflammatory and neuropathic pain. Here, we hypothesized that BDNF could be a trigger of some of these plastic changes. Our results demonstrate that BDNF is upregulated in the anterior cingulate cortex (ACC) and the primary sensory cortex (S1) in rats with inflammatory pain. Injections of recombinant BDNF (into the ACC) or a viral vector synthesizing BDNF (into the ACC or S1) triggered both neuronal hyperexcitability, as shown by elevated long-term potentiation, and sustained pain hypersensitivity. Finally, pharmacological blockade of BDNF-tropomyosin receptor kinase B (TrkB) signaling in the ACC, through local injection of cyclotraxin-B (a novel, highly potent, and selective TrkB antagonist) prevented neuronal hyperexcitability, the emergence of cold hypersensitivity, and passive avoidance behavior. These findings show that BDNF-dependent neuronal plasticity in the ACC, a structure known to be involved in the affective-emotional aspect of pain, is a key mechanism in the development and maintenance of the emotional aspect of chronic pain.

Molecular mechanisms underlying the enhanced analgesic effect of oxycodone compared to morphine in chemotherapy-induced neuropathic pain.

Oxycodone is a μ-opioid receptor agonist, used for the treatment of a large variety of painful disorders. Several studies have reported that oxycodone is a more potent pain reliever than morphine, and that it improves the quality of life of patients. However, the neurobiological mechanisms underlying the therapeutic action of these two opioids are only partially understood. The aim of this study was to define the molecular changes underlying the long-lasting analgesic effects of oxycodone and morphine in an animal model of peripheral neuropathy induced by a chemotherapic agent, vincristine. Using a behavioural approach, we show that oxycodone maintains an optimal analgesic effect after chronic treatment, whereas the effect of morphine dies down. In addition, using DNA microarray technology on dorsal root ganglia, we provide evidence that the long-term analgesic effect of oxycodone is due to an up-regulation in GABAB receptor expression in sensory neurons. These receptors are transported to their central terminals within the dorsal horn, and subsequently reinforce a presynaptic inhibition, since only the long-lasting (and not acute) anti-hyperalgesic effect of oxycodone was abolished by intrathecal administration of a GABAB receptor antagonist; in contrast, the morphine effect was unaffected. Our study demonstrates that the GABAB receptor is functionally required for the alleviating effect of oxycodone in neuropathic pain condition, thus providing new insight into the molecular mechanisms underlying the sustained analgesic action of oxycodone.

Activation-Dependent Subcellular Distribution Patterns of CB1 Cannabinoid Receptors in the Rat Forebrain

Chronic cannabinoid exposure results in tolerance due to region-specific desensitization and down-regulation of CB1 cannabinoid receptors (CB1Rs). For most G-protein-coupled receptors, internalization closely follows rapid desensitization as an important component of long-term down-regulation. However, in vivo patterns of CB1R internalization are not known. Here we investigate the subcellular redistribution of CB1Rs in the rat forebrain following activation by agonist CP55 940 or inhibition by antagonist/inverse agonist AM251. At steady state, CB1Rs are mainly localized to the cell membrane of preterminal axon shafts and, to a lesser degree, to synaptic terminals. A high proportion of CB1Rs is also localized to somatodendritic endosomes. Inhibition of basal activation by acute AM251 administration decreases the number of cell bodies containing CB1R-immunoreactive endosomes, suggesting that CB1Rs are permanently activated and internalized at steady state. On the contrary, acute agonist treatment induces rapid and important increase of endosomal CB1R immunolabeling, likely due to internalization and retrograde transport of axonal CB1Rs. Repeated agonist treatment is necessary to significantly reduce initially high levels of axonal CB1R labeling, in addition to increasing somatodendritic endosomal CB1R labeling in cholecystokinin-positive interneurons. This redistribution displays important region-specific differences; it is most pronounced in the neocortex and hippocampus and absent in basal ganglia.

Cortical effect of oxaliplatin associated with sustained neuropathic pain: Exacerbation of cortical activity and down-regulation of potassium channel expression in somatosensory cortex

Summary Down‐regulation of potassium channel in somatosensory cortex is a possible origin of cortical hyperexcitability in an oxaliplatin model. Abstract Oxaliplatin is a third‐generation platinum‐based chemotherapy drug that has gained importance in the treatment of advanced metastatic colorectal cancer. Its dose‐limiting side effect is the production of chronic peripheral neuropathy. Using a modified model of oxaliplatin‐induced sensory neuropathy, we investigated plastic changes at the cortical level as possible mechanisms underlying the chronicity of pain sensation in this model. Changes in gene expression were studied using DNA microarray which revealed that when oxaliplatin‐treated animals displayed clinical neuropathic pain symptoms, including mechanical and thermal hypersensitivity, approximately 900 were down‐regulated in the somatosensory cortex. Because of the known role of potassium channels in neuronal excitability, the study further focussed on the down‐regulation of these channels as the possible molecular origin of cortical hyperexcitability. Quantification of the magnitude of neuronal extracellular signal‐regulated kinase (ERK) phosphorylation in cortical neurons as a marker of neuronal activity revealed a 10‐fold increase induced by oxaliplatin treatment, suggesting that neurons of cortical areas involved in transmission of painful stimuli undergo a chronic cortical excitability. We further demonstrated, using cortical injection of lentiviral vector shRNA against Kv2.2, that down‐regulation of this potassium channel in naive animals induced a sustained thermal and mechanical hypersensitivity. In conclusion, although the detailed mechanisms leading to this cortical excitability are still unknown, our study demonstrated that a cortical down regulation of potassium channels could underlie pain chronicity in this model of chemotherapy‐induced neuropathic pain.

Structural and Molecular Alterations of Primary Afferent Fibres in the Spinal Dorsal Horn in Vincristine-Induced Neuropathy in Rat

Vincristine is one of the most common anti-cancer drug therapies administered for the treatment of many types of cancer. Its dose-limiting side effect is the emergence of peripheral neuropathy, resulting in chronic neuropathic pain in many patients. This study sought to understand the mechanisms underlying the development of neuropathic pain by vincristine-induced neurotoxicity. We focused on signs of functional changes and revealed that deep layers of the spinal cord (III–IV) experience increased neuronal activity both in the absence of peripheral stimulation and, as a result of tactile mechanical stimulations. These laminae and superficial laminae I–II were also subject to structural changes as evidenced by an increase in immunoreactivity of Piccolo, a marker of active presynaptic elements. Further investigations performed, using DNA microarray technology, describe a large number of genes differentially expressed in dorsal root ganglions and in the spinal dorsal horn after vincristine treatment. Our study describes an important list of genes differentially regulated by vincristine treatment that will be useful for future studies and brings forward evidence for molecular and anatomical modifications of large diameter sensory neurons terminating in deep dorsal horn laminae, which could participate in the development of tactile allodynia.

Role of NGF in Neuronal Plasticity in the Lateral Reticular Nucleus in Chronic Inflammatory Pain

In pathological states, repetitive inputs from the ascending pathways involved in the genesis and integration of nociception, leads to molecular, anatomical and electrophysiological adaptive changes of these pathways, contributing to the development of pain chronicity. In the past, neurotrophic factors have been implicated in neuronal plasticity in adult central nervous system. We have previously described plastic changes associated with the up-regulation of NGFs high affinity receptor, TrkA, in the spinoreticular pathway in a chronic inflammatory pain model of arthritis induced by complete Freunds adjuvant. The present study investigated the role of central NGF in the maintenance of inflammatory pain. Analysis of TrkA and NGF expression revealed that they are expressed in the medial thalamus and several reticular nuclei of the brain stem such as the lateral reticular nucleus (LRt) and not in pathways classically described to be involved in the sensory-discriminative aspect of pain such as the lateral thalamus. In addition NGF was over-expressed in the LRt, lateral thalamus and cortex of polyarthritic rats. Using micro-injection of an adenoviral vector synthesizing NGF (or green fluorescent protein) in the LRt of normal animals, we showed that increased NGF levels in the LRt leads to the development of mechanical hypersensitivity and increased nocifensive behavior following an inflammatory stimulus. These results suggest that, NGF acts centrally as a possible molecular inducer of synaptic plasticity in the LRt in conditions of chronic inflammatory pain.

Orofacial Neuropathic Pain Leads to a Hyporesponsive Barrel Cortex with Enhanced Structural Synaptic Plasticity.

Chronic pain is a long-lasting debilitating condition that is particularly difficult to treat due to the lack of identified underlying mechanisms. Although several key contributing processes have been described at the level of the spinal cord, very few studies have investigated the supraspinal mechanisms underlying chronic pain. Using a combination of approaches (cortical intrinsic imaging, immunohistochemical and behavioural analysis), our study aimed to decipher the nature of functional and structural changes in a mouse model of orofacial neuropathic pain, focusing on cortical areas involved in various pain components. Our results show that chronic neuropathic orofacial pain is associated with decreased haemodynamic responsiveness to whisker stimulation in the barrel field cortex. This reduced functional activation is likely due to the increased basal neuronal activity (measured indirectly using cFos and phospho-ERK immunoreactivity) observed in several cortical areas, including the contralateral barrel field, motor and cingulate cortices. In the same animals, immunohistochemical analysis of markers for active pre- or postsynaptic elements (Piccolo and phospho-Cofilin, respectively) revealed an increased immunofluorescence in deep cortical layers of the contralateral barrel field, motor and cingulate cortices. These results suggest that long-lasting orofacial neuropathic pain is associated with exacerbated neuronal activity and synaptic plasticity at the cortical level.