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Corneal pain is considered to be a core symptom of ocular surface disruption and inflammation. The management of this debilitating condition is still a therapeutic challenge. Recent evidence supports a role of the opioid system in the management of corneal nociception. However, the functional involvement of the mu opioid receptor (MOR) underlying this analgesic effect is not known. We first investigated the expression of the MOR in corneal nerve fibers and trigeminal ganglion (TG) neurons in control mice and a mouse model of corneal inflammatory pain. We then evaluated the anti-nociceptive and electrophysiological effects of DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin), a MOR-selective ligand. MOR immunoreactivity was detected in corneal nerve fibers and primary afferent neurons of the ophthalmic branch of the TG of naive mice. MOR expression was significantly higher in both structures under conditions of inflammatory corneal pain. Topical ocular administration of DAMGO strongly reduced both the mechanical (von Frey) and chemical (capsaicin) corneal hypersensitivity associated with inflammatory ocular pain. Repeated instillations of DAMGO also markedly reversed the elevated spontaneous activity of the ciliary nerve and responsiveness of corneal polymodal nociceptors that were observed in mice with corneal pain. Finally, these DAMGO-induced behavioral and electrophysiological responses were totally blunted by the topical application of naloxone methiodide, an opioid receptor antagonist. Overall, these results provide evidence that topical pharmacological MOR activation may constitute a therapeutic target for the treatment of corneal pain and improve corneal nerve function to alleviate chronic pain., This work was supported by the Sorbonne Université and the Institut National de la Santé et de la Recherche Médicale, the ANR, LabEx LIFESENSES (ANR-10-LABX-65), and IHU FOReSIGHT (ANR-18-IAHU-01). Fanny Joubert was supported by a Fondation de France post-doc fellowship grant. Adrian Guerrero Moreno was funded by a H2020-MSCA-ETN program (IT-DED3) (Grant Agreement 765608).

[Background]: Ocular surface diseases are among the most frequent ocular pathologies. Ocular pain following corneal injury is frequently observed in clinic. Corneal sensory innervation is supplied by ciliary nerves derived from ophthalmic division of the trigeminal ganglion., [Methods & Results]: Extracellular activity of the mouse ciliary nerve was first used to investigate the corneal responsiveness to chemical, mechanical and thermal stimulations in order to specifically study the responses of polymodal nociceptors, mechano‐nociceptors and cold thermoreceptor in a control cornea. Then, in two models of corneal injury (repeated instillations of 0.02% benzalkonium chloride and corneal scraping), we first measured the corneal sensitivity to chemical (eye‐wiping test) and mechanical (von Frey filaments) stimulation. Thereafter, we evaluated whether these corneal injuries modified the spontaneous and chemical stimulation‐evoked activity of the ciliary nerve. Both models of injury induced a significant corneal chemical hypersensitivity correlated with an increase of the spontaneous activity of the ciliary nerve and a faster response of the ciliary nerve after a chemical stimulation., [Conclusions]: Overall, this study provides new insights into the functional aspects of corneal nerve fibre activity in mice after corneal injury. The increase in ciliary nerve activity may thus contribute to the development of ocular pain after corneal damage., [Significance]: This study highlights the parallel increase in ciliary nerve activity and corneal sensitivity after corneal injury in mice. The strategy of combining ex vivo electrophysiological recordings of the ciliary nerve in mice and corneal sensitivity measurements therefore helps to uncover the functional aspects of corneal pain., This work was supported by Sorbonne Université and the Institut National de la Santé et de la Recherche Médicale, LABEX (LBX3‐OPANI). FJ received a Fondation de France post‐doc fellowship grant.

BackgroundDry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches.MethodsA mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21.ResultsWe observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1β), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines (IL-6, TNFα, IL-1β, and CCL2), iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1.ConclusionsOverall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain.

Background Central alveolar hypoventilation syndromes (CHS) encompass neurorespiratory diseases resulting from congenital or acquired neurological disorders. Hypercapnia, acidosis, and hypoxemia resulting from CHS negatively affect physiological functions and can be lifethreatening. To date, the absence of pharmacological treatment implies that the patients must receive assisted ventilation throughout their lives. Objective To highlight the relevance of determining conditions in which using gonane synthetic progestins could be of potential clinical interest for the treatment of CHS. Methods The mechanisms by which gonanes modulate the respiratory drive were put into the context of those established for natural progesterone and other synthetic progestins. Results The clinical benefits of synthetic progestins to treat respiratory diseases are mixed with either positive outcomes or no improvement. A benefit for CHS patients has only recently been proposed. We incidentally observed restoration of CO2 chemosensitivity, the functional deficit of this disease, in two adult CHS women by desogestrel, a gonane progestin, used for contraception. This effect was not observed by another group, studying a single patient. These contradictory findings are probably due to the complex nature of the action of desogestrel on breathing and led us to carry out mechanistic studies in rodents. Our results show that desogestrel influences the respiratory command by modulating the GABAA and NMDA signaling in the respiratory network, medullary serotoninergic systems, and supramedullary areas. Conclusion Gonanes show promise for improving ventilation of CHS patients, although the conditions of their use need to be better understood.

International audience; We mapped and characterized changes in the activity of brainstem cell groups under hypoxia in one-day-old newborn mice, an animal model in which the central nervous system at birth is particularly immature. The classical biphasic respiratory response characterized by transient hyperventilation, followed by severe ventilation decline, was associated with increased c-FOS immunoreactivity in brainstem cell groups: the nucleus of the solitary tract, ventral reticular nucleus of the medulla, retrotrapezoid/parafacial region, parapyramidal group, raphe magnus nucleus, lateral, and medial parabrachial nucleus, and dorsal subcoeruleus nucleus. In contrast, the hypoglossal nucleus displayed decreased c-FOS immunoreactivity. There were fewer or no activated catecholaminergic cells activated in the medulla oblongata, whereas ∼45% of the c-FOS-positive cells in the dorsal subcoeruleus were co-labeled. Approximately 30% of the c-FOS-positive cells in the parapyramidal group were serotoninergic, whereas only a small portion were labeled for serotonin in the raphe magnus nucleus. None of the c-FOS-positive cells in the retrotrapezoid/parafacial region were co-labeled for PHOX2B. Thus, the hypoxia-activated brainstem neuronal network of one-day-old mice is characterized by (i) the activation of catecholaminergic cells of the dorsal subcoeruleus nucleus, a structure implicated in the strong depressive pontine influence previously reported in the fetus but not in newborns, (ii) the weak activation of catecholaminergic cells of the ventral reticular nucleus of the medulla, an area involved in hypoxic hyperventilation, and (iii) the absence of PHOX2B-positive cells activated in the retrotrapezoid/parafacial region. Based on these results, one-day-old mice could highlight characteristics for modeling the breathing network of premature infants.

International audience; Central congenital hypoventilation syndrome (CCHS) is a neurorespiratory disease characterized by life-threatening sleep-related hypoventilation involving an alteration of CO2/H+ chemosensitivity. Incidental findings have suggested that desogestrel may allow recovery of the ventilatory response to CO2. The effects of desogestrel on resting ventilation have not been reported. This study was designed to test the hypothesis that desogestrel strengthens baseline ventilation by analyzing the ventilation of CCHS patients. Rodent models were used in order to determine the mechanisms involved. Ventilation in CCHS patients was measured with a pneumotachometer. In mice, ventilatory neural activity was recorded from ex vivo medullary-spinal cord preparations, ventilation was measured by plethysmography and c-fos expression was studied in medullary respiratory nuclei. Desogestrel increased baseline respiratory frequency of CCHS patients leading to a decrease in their PETCO2. In medullary spinal-cord preparations or in vivo mice, the metabolite of desogestrel, etonogestrel, induced an increase in respiratory frequency that necessitated the functioning of serotoninergic systems, and modulated GABAA and NMDA ventilatory regulations. c-FOS analysis showed the involvement of medullary respiratory groups of cell including serotoninergic neurons of the raphe pallidus and raphe obscurus nuclei that seem to play a key role. Thus, desogestrel may improve resting ventilation in CCHS patients by a stimulant effect on baseline respiratory frequency. Our data open up clinical perspectives based on the combination of this progestin with serotoninergic drugs to enhance ventilation in CCHS patients.