Molecular and behavioral exploration of orofacial hypersensitivity in a model of oxaliplatin-chemotherapy induced acute neuropathy Laura POUPON, 1 ère année de thèse U1107 – Pharmacologie fondamentale et clinique de la douleur Directeur : Pr. A. Eschalier 9ème Symposium National du Réseau Recherche sur la Douleur 22 et 23 mars 2013

Oxaliplatin chemotherapy-induced neuropathy Therapeutic effects Adverse effects Used in colorectal cancer chemotherapy Inhibits DNA replication by platinum adduct formation 90% of patients develop acute neuropathy Cold hypersensitivity Numbness at both extracephalic and cephalic territories Dose reduction to limit neuropathic symptoms Cephalic territory Extracephalic territory Oxaliplatin (Eloxatin®) Platinum-based antineoplasic

DRG Cold hyperalgesia Mechanical hypersensitivity Existing extracephalic results in mice Mice model Clinical symptoms Descoeur et al., 2011

Mice ion channels transcriptional expression Mice ion channels transcriptional expression Existing extracephalic results in mice DRG Descoeur et al., 2011 Correlation between oxaliplatin-induced cold hypersensitivity and remodelling of ion channel expression in nociceptors Clinical symptoms DRG

Differences between cephalic and extracephalic sensory systems TG C-fibers Sensory systems A-fibers Caudalis Cephalic ? DRG Extracephalic

Aim of the study Development of adapted orofacial behavioral tests Assessment of ion channel transcriptional plasticity at the TG level Genetic or pharmacological validation of ion channels as targets of interest ? Are there behavioral and molecular differences between the cephalic and the extracephalic territories in a murine model of oxaliplatin chemotherapy-induced neuropathy ?

Material & Methods Mice model Single i.p. injection of oxaliplatin (6mg/kg) Peak pain behavior at D4 Daily measures of pain scores from D0 to D4 Day D0 D1 D2 D3 D4 Behavioral tests Caudalis -TG HIC (pERK) qPCR (ion channels)

Oral cold perception in WT mice * * Water consumption (mL) Oxaliplatin VehiclepERK Cold water aversion test Vehicle Oxaliplatin 6 mg/kg Oral cold Hypersensitivity D4 - Caudalis D0 D1 D2 D3 D4 3h 4 - 6°C

Cutaneous cold and Mechanical perception in WT mice Score Vehicle Oxaliplatin 6 mg/kg *** * * Brush test Response to dynamic mechanical stimulation 5 consecutive strokes Grooming (s) *** * Vehicle Oxaliplatin 6 mg/kg Acetone test Daily measure of grooming behavior On the upper lip area skin surface Cutaneous cold Hypersensitivity Mechanical hypersensitivity

Ion channels plasticity in DRG and TG qPCR Extracephalic (DRG) Cephalic (TG) Vehicle Oxaliplatin

Cold sensors : TRPM8 and TRPA1 Grooming (s) Score NS ** Vehicle Oxaliplatin 6 mg/kg TRPM8 -/- mice Oxaliplatin fail to induce cold hypersensitivity and preserve mechanical pain symptoms in TRPM8 null mice Vehicle TRPA1 Antagonist (HC030031) Grooming (s) TRPA1 implication in cold hypersensitivity WT mice Score ** NS qPCR Acetone test Brush test * ** NS * ** Grooming (s) Score

HCN channels : ** Score NS NaCl Ivabradine NaCl Ivabradine Vehicle Oxaliplatin Acetone test Brush test Grooming (s) ** * Ivabradine (Procoralan®) Non-specific HCN antagonist Reversion of cold hypersensitivity No effects on mechanical sensibility Oxaliplatin Vehicle HCN1 Xx40

Conclusion CEPHALIC cold and mechanical hypersensitivity induced by oxaliplatin Differencies between cephalic and extracephalic territories at both molecular and pharmacological levels Extracephalic (DRG)Cephalic (TG) TRPM TRPA HCN1 - HCN

Perspectives Impact of oxaliplatin on functional TG neurons? Ca 2+ imaging Involvement of K2P TREK and TRAAK channels? Use of other KO mice (TREK2, TREK1-TRAAK) Cephalic and extracephalic behavioral, molecular & pharmacological consequences of repeated oxaliplatin injections Short term Long term

Acknowledgements Project realized in association with Institut de génomique fonctionnelle (Montpellier) Anne PIZZOCCARO, Emmanuel BOURINET Inserm U1107 (Clermont Ferrand) Neurobiologie de la douleur trigéminale Amélie DESCHEMEAKER, Radhouane DALLEL Laboratoire de pharmacologie fondamentale et clinique de la douleur Vanessa PEREIRA, Maryse CHALUS, Alain ESCHALIER, Jérôme BUSSEROLLES Thank you for your attention

Vandewauw et al., 2013 Channels expression TRPA1 TRPM8

Cold water aversion test Results at 20°C 20 °C Vehicle x20 Oxaliplatin pERK

HCN Channels Postea et al., 2011