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Background: Glaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model., Methods: Rat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b + , CD68 + , and CD11b + /CD68 + cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR., Results: Y-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1 + /CD68 + cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b + , CD68 + , and CD11b + /CD68 + cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1β, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects., Conclusion: Y-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma., (© 2024. The Author(s).)

Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. Corneal pain, as one of DED's main symptoms, has gained recognition due to its increasing prevalence, morbidity, and the resulting social burden. The cornea is the most innervated tissue in the body, and the maintenance of corneal integrity relies on a rich density of nociceptors, such as polymodal nociceptor neurons, cold thermoreceptor neurons, and mechano-nociceptor neurons. Their sensory responses to different stimulating forces are linked to the specific expression of transient receptor potential (TRP) channels. TRP channels are a group of unique ion channels that play important roles as cellular sensors for various stimuli. These channels are nonselective cation channels with variable Ca 2+ selectivity. TRP homologs are a superfamily of 28 different members that are subdivided into 7 different subfamilies based on differences in sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells, where they affect various stress-induced regulatory responses essential for normal vision maintenance. This article reviews the current knowledge about the expression, function, and regulation of TRPs in ocular surface tissues. We also describe their implication in DED and ocular pain. These findings contribute to evidence suggesting that drug-targeting TRP channels may be of therapeutic benefit in the clinical setting of ocular pain., Competing Interests: The company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The cornea, an anterior ocular tissue that notably serves to protect the eye from external insults and refract light, requires constant epithelium renewal and efficient healing following injury to maintain ocular homeostasis. Although several key cell populations and molecular pathways implicated in corneal wound healing have already been thoroughly investigated, insufficient/impaired or excessive corneal wound healing remains a major clinical issue in ophthalmology, and new avenues of research are still needed to further improve corneal wound healing. Because of its implication in numerous cellular/tissular homeostatic processes and oxidative stress, there is growing evidence of the role of Hedgehog signaling pathway in physiological and pathological corneal wound healing. Reviewing current scientific evidence, Hedgehog signaling and its effectors participate in corneal wound healing mainly at the level of the corneal and limbal epithelium, where Sonic Hedgehog-mediated signaling promotes limbal stem cell proliferation and corneal epithelial cell proliferation and migration following corneal injury. Hedgehog signaling could also participate in corneal epithelial barrier homeostasis and in pathological corneal healing such as corneal injury-related neovascularization. By gaining a better understanding of the role of this double-edged sword in physiological and pathological corneal wound healing, fascinating new research avenues and therapeutic strategies will undoubtedly emerge.

Background: Glaucoma is a blinding degenerative neuropathy in which the death of retinal ganglion cells (RGCs) causes progressive loss of visual field and eventually vision. Neuroinflammation appears to be a key event in the progression and spread of this disease. Thus, microglial immunomodulation represents a promising therapeutic approach in which mesenchymal stem cells (MSCs) might play a crucial role. Their neuroprotective and regenerative potentials have already raised hope in animal models. Yet no definitive treatment has been developed, and some safety concerns have been reported in human trials. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model., Methods: Labeled rat bone marrow MSCs were placed in coculture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a, and NeuN markers. Gliosis and retinal microglial activation were measured by using GFAP, CD68, and ITGAM mRNA quantification and GFAP, CD68, and Iba1 immunofluorescence stainings. We also analyzed the mRNA expression of both 'M1' or classically activated state inflammatory cytokines (TNFα, IL1β, and IL6), and 'M2' or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6)., Results: The number of RGCs was significantly higher in retinal explants cultured with MSCs compared to the control group at Day 7 following the optic nerve axotomy. Retinal explants cultured with MSCs showed a decrease in mRNA markers of gliosis and microglial activations, and immunostainings revealed that GFAP, Iba1, and CD68 were limited to the inner layers of the retina compared to controls in which microglial activation was observed throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in presence of MSCs, with an increase in fibronectin labeling at the surface of the explant corresponding to an epiretinal membrane-like phenotype., Conclusion: Using an ex vivo neuroretina model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling., (© 2022. The Author(s).)

The trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and its dysfunction leads to intraocular pressure elevation, which is one of the main risk factors of glaucoma. We aimed to compare the effects on cytoskeleton organization and extracellular matrix (ECM) of latanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF-β2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). The TGF-β2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h. The expression of alpha-smooth muscle actin (αSMA) and fibronectin (FN), and phosphorylation of the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescence and Western blot. The architectural modifications were studied in a Matrigel TM 3D culture. TGF-β2 increased the expression of αSMA and FN in pHTMC and modified the cytoskeleton with cross-linked actin network formation. LT did not alter the expression of αSMA but decreased FN deposition. The ROCKi decreased TGF-β2-induced αSMA and FN expression, as well as MLC-P and Cofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary 3D study, pHTMC organized in a mesh conformation showed the widening of the TM under the effect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM, with fibronectin deposition and overexpression, TGF-β2 reproduced the trabecular degeneration described in glaucoma. The ROCKi was able to reverse the TGF-β2-induced cytoskeletal and ECM rearrangements. LT loosened the extracellular matrix but had no action on the stress fibers.

The in-depth knowledge of lipid biological functions needs a comprehensive structural annotation including a method to locate fatty acid unsaturations, which remains a thorny problem. For this purpose, we have associated Grubbs' cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry to locate double bond positions in lipid species. The pretreatment of lipid-containing samples by Grubbs' catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains of phospholipids, glycerolipids, and sphingolipids. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts of human corneal epithelial (HCE) cell line allowing to pinpoint double bond of more than 90 species. This method has also been useful to investigate the lipid homeostasis alteration in an in vitro model of corneal toxicity, i.e., HCE cells incubated with benzalkonium chloride. The association of cross-metathesis and tandem mass spectrometry appears suitable to locate double bond positions in lipids involved in relevant biological processes., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Pain is reported as the leading cause of disability in the common forms of inflammatory arthritis conditions. Acting as a key player in nociceptive processing, neuroinflammation, and neuron-glia communication, the chemokine CCL2/CCR2 axis holds great promise for controlling chronic painful arthritis. Here, we investigated how the CCL2/CCR2 system in the dorsal root ganglion (DRG) contributes to the peripheral inflammatory pain sensitization., Methods: Repeated intrathecal (i.t.) administration of the CCR2 antagonist, INCB3344 was tested for its ability to reverse the nociceptive-related behaviors in the tonic formalin and complete Freund's adjuvant (CFA) inflammatory models. We further determined by qPCR the expression of CCL2/CCR2, SP and CGRP in DRG neurons from CFA-treated rats. Using DRG explants, acutely dissociated primary sensory neurons and calcium mobilization assay, we also assessed the release of CCL2 and sensitization of nociceptors. Finally, we examined by immunohistochemistry following nerve ligation the axonal transport of CCL2, SP, and CGRP from the sciatic nerve of CFA-treated rats., Results: We first found that CFA-induced paw edema provoked an increase in CCL2/CCR2 and SP expression in ipsilateral DRGs, which was decreased after INCB3344 treatment. This upregulation in pronociceptive neuromodulators was accompanied by an enhanced nociceptive neuron excitability on days 3 and 10 post-CFA, as revealed by the CCR2-dependent increase in intracellular calcium mobilization following CCL2 stimulation. In DRG explants, we further demonstrated that the release of CCL2 was increased following peripheral inflammation. Finally, the excitation of nociceptors following peripheral inflammation stimulated the anterograde transport of SP at their peripheral nerve terminals. Importantly, blockade of CCR2 reduced sensory neuron excitability by limiting the calcium mobilization and subsequently decreased peripheral transport of SP towards the periphery. Finally, pharmacological inhibition of CCR2 reversed the pronociceptive action of CCL2 in rats receiving formalin injection and significantly reduced the neurogenic inflammation as well as the stimuli-evoked and movement-evoked nociceptive behaviors in CFA-treated rats., Conclusions: Our results provide significant mechanistic insights into the role of CCL2/CCR2 within the DRG in the development of peripheral inflammation, nociceptor sensitization, and pain hypersensitivity. We further unveil the therapeutic potential of targeting CCR2 for the treatment of painful inflammatory disorders.

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-Ala 2 ,N-Me-Phe 4 ,Gly 5 -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., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.

Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MPs) of the peripheral nervous system (PNS) are the main cellular agent controlling this process. Some evidence suggests that resident PNS-MPs along with MPs of hematogenous origin may be involved, but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporter mice and coherent anti-Stokes Raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatiotemporal choreography of resident and recently recruited MPs after injury and unveiled distinct functions of these subsets, with recruited MPs being responsible for efficient myelin stripping and clearance and resident MPs being involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Boissonnas et al.)

Detergent chemicals, widely used in household products, in pharmaceutical, medical, cosmetic and industrial fields, have been linked to side effects and involved in several eye diseases. On the ocular surface, detergents can interfere with the corneal epithelium, the most superficial layer of the cornea, representing a line of defence against external aggression. Despite its major role in numerous biological functions, there is still little data regarding disruption of lipid homeostasis induced by ocular irritants. To this purpose, a lipidomic analysis using UPLC-HRMS/MS-ESI ± was performed on human corneal epithelial (HCE) cells incubated with three widely known ocular irritants: benzalkonium chloride (BAK), sodium lauryl sulfate (SLS) and Triton X-100 (TXT). We found that these ocular irritants lead to a profound modification of the HCE cell lipidome. Indeed, the cell content of ceramide species increased widely while plasmalogens containing polyunsaturated fatty acid species, especially docosahexaenoic acids, decreased. Furthermore, these irritants upregulated the activity of phospholipase A 2 . The present study demonstrates that BAK, SLS and TXT induced disruption of the cell lipid homeostasis, highlighting that lipids mediate inflammatory and cell death processes induced by detergents in the cornea. Lipidomics may thus be regarded as a valuable tool to investigate new markers of corneal damage., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Dry eye disease (DED) is a multifactorial chronic inflammatory disease of the ocular surface characterized by tear film instability, hyperosmolarity, cell damage and inflammation. Hyperosmolarity is strongly established as the core mechanism of the DED. Benzalkonium chloride (BAK) - a quaternary ammonium salt commonly used in eye drops for its microbicidal properties - is well known to favor the onset of DED. Currently, little data are available regarding lipid metabolism alteration in ocular surface epithelial cells in the course of DED. Our aim was to explore the effects of benzalkonium chloride or hyperosmolarity exposure on the human corneal epithelial (HCE) cell lipidome, two different conditions used as in vitro models of DED. For this purpose, we performed a lipidomic analysis using UPLC-HRMS-ESI+/-. Our results demonstrated that BAK or hyperosmolarity induced important modifications in HCE lipidome including major changes in sphingolipids, glycerolipids and glycerophospholipids. For both exposures, an increase in ceramide was especially exhibited. Hyperosmolarity specifically induced triglyceride accumulation resulting in lipid droplet formation. Conversely, BAK induced an increase in lysophospholipids and a decrease in phospholipids. This lipidomic study highlights the lipid changes involved in inflammatory responses following BAK or hyperosmolarity exposures. Thereby, lipid research appears of great interest, as it could lead to the discovery of new biomarkers and therapeutic targets for the diagnosis and treatment of dry eye disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Annotation of lipids in untargeted lipidomic analysis remains challenging and a systematic approach needs to be developed to organize important datasets with the help of bioinformatic tools. For this purpose, we combined tandem mass spectrometry-based molecular networking with retention time (t R ) prediction to annotate phospholipid and sphingolipid species. Sixty-five standard compounds were used to establish the fragmentation rules of each lipid class studied and to define the parameters governing their chromatographic behavior. Molecular networks (MNs) were generated through the GNPS platform using a lipid standards mixture and applied to lipidomic study of an in vitro model of dry eye disease, i.e. , human corneal epithelial (HCE) cells exposed to hyperosmolarity (HO). These MNs led to the annotation of more than 150 unique phospholipid and sphingolipid species in the HCE cells. This annotation was reinforced by comparing theoretical to experimental t R values. This lipidomic study highlighted changes in 54 lipids following HO exposure of corneal cells, some of them being involved in inflammatory responses. The MN approach coupled to t R prediction thus appears as a suitable and robust tool for the discovery of lipids involved in relevant biological processes.

Glaucoma is one of the leading causes of irreversible blindness in the world and remains a major public health problem. To date, incomplete knowledge of this disease's pathophysiology has resulted in current therapies (pharmaceutical or surgical) unfortunately having only a slowing effect on disease progression. Recent research suggests that glaucomatous optic neuropathy is a disease that shares common neuroinflammatory mechanisms with "classical" neurodegenerative pathologies. In addition to the death of retinal ganglion cells (RGCs), neuroinflammation appears to be a key element in the progression and spread of this disease. Indeed, early reactivity of glial cells has been observed in the retina, but also in the central visual pathways of glaucoma patients and in preclinical models of ocular hypertension. Moreover, neuronal lesions are not limited to retinal structure, but also occur in central visual pathways. This review summarizes and puts into perspective the experimental and clinical data obtained to date to highlight the need to develop neuroprotective and immunomodulatory therapies to prevent blindness in glaucoma patients., Competing Interests: “The authors declare no conflict of interest.”

Background: Dry 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., Methods: A 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., Results: We 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., Conclusions: Overall, 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.

Photophobia may arise from various causes and frequently accompanies numerous ocular diseases. In modern highly illuminated world, complaints about greater photosensitivity to blue light increasingly appear. However, the pathophysiology of photophobia is still debated. In the present work, we investigated in vivo the role of various neural pathways potentially implicated in blue-light aversion. Moreover, we studied the light-induced neuroinflammatory processes on the ocular surface and in the trigeminal pathways. Adult male C57BL/6J mice were exposed either to blue (400-500 nm) or to yellow (530-710 nm) LED light (3 h, 6 mW/cm 2 ). Photosensitivity was measured as the time spent in dark or illuminated parts of the cage. Pharmacological treatments were applied: topical instillation of atropine, pilocarpine or oxybuprocaine, intravitreal injection of lidocaine, norepinephrine or "blocker" of the visual photoreceptor transmission, and intraperitoneal injection of a melanopsin antagonist. Clinical evaluations (ocular surface state, corneal mechanical sensitivity and tear quantity) were performed directly after exposure to light and after 3 days of recovery in standard light conditions. Trigeminal ganglia (TGs), brainstems and retinas were dissected out and conditioned for analyses. Mice demonstrated strong aversion to blue but not to yellow light. The only drug that significantly decreased the blue-light aversion was the intraperitoneally injected melanopsin antagonist. After blue-light exposure, dry-eye-related inflammatory signs were observed, notably after 3 days of recovery. In the retina, we observed the increased immunoreactivity for GFAP, ATF3, and Iba1; these data were corroborated by RT-qPCR. Moreover, retinal visual and non-visual photopigments distribution was altered. In the trigeminal pathway, we detected the increased mRNA expression of cFOS and ATF3 as well as alterations in cytokines' levels. Thus, the wavelength-dependent light aversion was mainly mediated by melanopsin-containing cells, most likely in the retina. Other potential pathways of light reception were also discussed. The phototoxic message was transmitted to the trigeminal system, inducing both inflammation at the ocular surface and stress in the retina. Further investigations of retina-TG connections are needed.

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., (© 2018 European Pain Federation - EFIC®.)

Aims: The ocular surface is the very first barrier between the visual system and external environment. It protects the eye from the exposure to various light sources that significantly emit in blue spectrum. However, the impact of blue light on the ocular surface has been poorly explored so far. In this study, we investigated in vitro the phototoxicity of blue light illumination in human epithelial cells of the ocular surface. We worked either in basal conditions or under hyperosmolar stress, in order to mimic dry eye disease (DED) that is the most common disease involving the ocular surface., Results: Corneal and conjunctival epithelial cells suffered the most from violet-blue light but also from longer-wave blue light. Exposure to blue wavebands significantly decreased cellular viability, impacted on cellular morphology and provoked reactive oxygen species (ROS) over-production. Conjunctival epithelial cell line had a greater photosensitivity than the corneal epithelial one. Hyperosmolar stress potentiated the blue light phototoxicity, increasing inflammation, altering mitochondrial membrane potential, and triggering the glutathione-based antioxidant system., Innovation: In human epithelial corneal and conjunctival cells of the ocular surface, we demonstrated the harmful impact of blue light on viability, redox state and inflammation processes, which was modified by hyperosmolarity., Conclusion: Blue light induced cell death and significant ROS production, and altered the expression of inflammatory genes and operation of the cellular defensive system. We established for the first time that hyperosmolar stress impacted phototoxicity, further suggesting that DED patients might be more sensitive to blue light ocular toxicity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Academic and industrial research has brought new insights into the pathogenesis of glaucoma, aiming at identifying and targeting specific mechanisms to improve our current therapeutic strategy. Retinal neurodegeneration is still the main focus, whether in terms of extrinsic factors such as neurotrophin deprivation, glutamate toxicity, vascular deficiency and neuro-inflammation from glial cells, or in terms of retinal ganglion cell intrinsic sensibility to proapoptotic signals. However, glaucoma is not solely a retinal disease but also involves retinal and trabecular meshwork degeneration, extending into and/or even originating from the brain. The present review summarizes our current knowledge of key mechanisms involved in glaucoma degeneration, focusing on the direction of current research towards the future of glaucoma therapy., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Chemokines and opioids are important regulators of immune, inflammatory and neuronal responses in peripheral and central pain pathways. Recent studies have provided insights into the functional interactions between chemokine receptors and opioid receptors, and their role in pain modulation. In this Progress article, we discuss how crosstalk between these two systems might provide a molecular and cellular framework for the development of novel analgesic therapies for the management of acute and/or chronic pain.

Background: Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aβ-fiber excitability following persistent inflammation., Methods: Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats., Results: Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aβ-fibers contributes to inflammatory pain., Conclusions: Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.

Chronic neuropathic pain has become a real social issue, due to the difficulty of its treatment and by the major impairment to quality of life that it causes in every day behavior. Understanding neurobiological basis and pathophysiological causes of diverse painful syndromes constantly evolves and reports the complexity of its mechanisms. Unfortunately this complexity makes it difficult to discover effective treatments against chronic pain syndromes, in particular as regards peripheral neuropathic pains. Recent studies reveal that, during chronic peripheral neuropathy, inflammatory mediators (in particular chemokines), besides their implications in the modulation of nociceptive messages and central neuroinflammatory mechanisms, play a critical role in the orchestration of the immune response induced by a peripheral nerve lesion. In this review, after a brief introduction about chemokines and their role in neuromodulation of the nociceptive message, we will attempt to define their functions and implications in the immune response associated to peripheral neuropathies. Thus, perfectly understanding the molecular and cellular communications between the nervous system and the immune system will be useful for the future development of novel and innovative therapeutic strategies against these highly disabling pathologies., (© Société de Biologie, 2014.)

Chemokines - chemotactic cytokines - are small secreted proteins that attract and activate immune and non-immune cells in vitro and in vivo. It has been suggested that chemokines and their receptors play a role in the central nervous system (CNS), in addition to their well established role in the immune system. We focus here on three chemokines-CXCL12 (C-X-C motif ligand 12), CCL2 (C-C motif ligand 2), and CX3CL1 (C-X-3C motif ligand 1) - and their principal receptors - CXCR4 (C-X-C motif receptor 4), CCR2 (C-C motif receptor 2) and CX3CR1 (C-X-3C motif receptor 1), respectively. We first introduce the classification of chemokines and their G-protein coupled receptors and the main signaling pathways triggered by receptor activation. We then discuss the cellular distribution of CXCL12/CXCR4, CCL2/CCR2 and CX3CL1/CX3CR1 in adult brain and the neurotransmission and neuromodulation effects controlled by these chemokines in the adult CNS. Changes in the expression of CXCL12, CCL2 and CX3CL1 and their respective receptors are also increasingly being implicated in the pathogenesis of CNS disorders, such as Alzheimer's disease, Parkinson's disease, HIV-associated encephalopathy, stroke and multiple sclerosis, and are therefore plausible targets for future pharmacological intervention. The final section thus discusses the role of these chemokines in these pathophysiological states. In conclusion, the role of these chemokines in cellular communication may make it possible: (i) to identify new pathways of neuron-neuron, glia-glia or neuron-glia communications relevant to both normal brain function and neuroinflammatory and neurodegenerative diseases; (ii) to develop new therapeutic approaches for currently untreatable brain diseases., (Published by Elsevier Ltd.)

Recent observations suggest that besides their role in the immune system, chemokines have important functions in the brain. There is a great line of evidence to suggest that chemokines are a unique class of neurotransmitters/neuromodulators, which regulate many biological aspects as diverse as neurodevelopment, neuroinflammation and synaptic transmission. In physiopathological conditions, many chemokines are synthesized in activated astrocytes and microglial cells, suggesting their involvement in brain defense mechanisms. However, when evoking chemokine functions in the nervous system, it is important to make a distinction between resting conditions and various pathological states including inflammatory diseases, autoimmune or neurodegenerative disorders in which chemokine functions have been extensively studied. We illustrate here the emergent concept of the neuromodulatory/neurotransmitter activities of neurochemokines and their potential role as a regulatory alarm system and as a group of messenger molecules for the crosstalk between neurons and cells from their surrounding microenvironment. In this deliberately challenging review, we provide novel hypotheses on the role of these subtle messenger molecules in brain functions leading to the evidence that previous dogmas concerning chemokines should be reconsidered., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

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.

Chemokines are small secreted proteins that chemoattract and activate immune and non-immune cells. Their role in the immune system is well-known, and it has recently been suggested that they may also play a role in the central nervous system (CNS). Indeed, they do not only act as immunoinflammatory mediators in the brain but they also act as potential modulators in neurotransmission. Although we are only beginning to be aware of the implication of chemokines in neuroendocrine functions, this review aims at summarizing what is known in that booming field of research. First we describe the expression of chemokines and their receptors in the CNS with a focus on the hypothalamo-pituitary system. Secondly, we present what is known on some chemokines in the regulation of neuroendocrine functions such as cell migration, stress, thermoregulation, drinking and feeding as well as anterior pituitary functions. We suggest that chemokines provide a fine modulatory tuning system of neuroendocrine regulations., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chemokines are small proteins belonging to the family of inflammatory cytokines whose main property is to attract and activate immune cells. Chemokines produce their biological activity by interacting with receptors belonging to the family of G protein-coupled receptors (GPCR). It is now clearly demonstrated that, under non-pathological conditions, chemokines and their receptors are constitutively expressed in the nervous system. Exploration of the implication of chemokines and their receptors in pain is a rapidly expanding domain of research. Recent results demonstrate that chemokines are active as modulators of nociceptive information both in the peripheral and central nervous system. After a brief recall of the chemokinergic system, we will discuss recent work on CCL2, a chemo-attractive cytokine which binds to the CCR2 receptor. There is a large body of data supporting the hypothesis that the CCL2 chemokine, released in the dorsal horn of the spinal cord, could have a direct effect on the transmission of nociceptive information and thus participate in neuronal adaptation and probably in the plasticity of glial cells and attractivity of immune cells observed during the installation and development of chronic pain arising from an inflammatory and/or neuropathic mechanism., (© Société de Biologie, 2011.)

Previous neuroanatomical studies realized in our team allowed us to demonstrate the neuronal and glial expression of various chemokines and their receptors in central dopaminergic (DA) pathways. In the light of these original observations, we questioned the role of chemokines on the physiology of DA neuron and on the neurodegenerative process in the DA nigro-striatal pathway, which characterizes Parkinson's disease. We focused our attention on two particular chemokines, the Stromal cell-Derived Factor-1 (SDF-1/CXCL12) and the Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) and their cognate receptors CXCR4 and CCR2, as they are expressed constitutively in nearly all DA mesencephalic neurons. We demonstrated, by using in vivo and in vitro approaches, that SDF-1 and MCP-1 can modulate DA neurotransmission in the nigro-striatal pathway, modifying the electrophysiological state of the neuron and DA release, through their cognate receptors. These effects are produced through N-type high voltage-activated calcium currents for SDF-1 and potassium channels for MCP-1. We then discuss the possible implication of SDF-1 and its derivative SDF-1(5-67) in DA neurodegeneration., (© Société de Biologie, 2011.)

Chemokines are not only mediators of the immune system and expressed in inflammatory situations. They are also constitutively expressed in the brain in both glial cells and neurons. Several recent evidence suggest that they can have a neurotransmitter/neuromodulatory role on brain functions similar to several neuropeptides reported so far. The aim of this short review is to illustrate that point using two chemokine systems, SDF-1/CXCL12 and its receptor CXCR4 and MCP-1/CCL2 and its receptor CCR2.

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.

Chemokines play a key role in inflammation. They are expressed not only in neuroinflammatory conditions, but also constitutively by different cell types, including neurons in the normal brain, suggesting that they may act as modulators of neuronal functions. Here, we investigated a possible neuroendocrine role of the chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12. We demonstrated the colocalization of SDF-1 and its receptor CXCR4 with arginine vasopressin (AVP) in the magnocellular neurons of the supraoptic nucleus (SON) and the paraventricular hypothalamic nucleus and on AVP projections to the neurohypophysis. Electrophysiological recordings of SON neurons demonstrated that SDF-1 affects the electrical activity of AVP neurons through CXCR4, resulting in changes in AVP release. We observed that SDF-1 can blunt the autoregulation of AVP release in vitro and counteract angiotensin II-induced plasma AVP release in vivo. Furthermore, a short-term physiological increase in AVP release induced by enhanced plasma osmolarity, which was produced by the administration of 1 M NaCl i.p., was similarly blocked by central injection of SDF-1 through CXCR4. A change in water balance by long-term salt loading induced a decrease in both SDF-1 and CXCR4 parallel to that of AVP immunostaining in SON. From these data, we demonstrate that chemokine actions in the brain are not restricted to inflammatory processes. We propose to add to the known autoregulation of AVP on its own neurons, a second autocrine system induced by SDF-1 able to modulate central AVP neuronal activity and release.

Chemokines and their receptors are well described in the immune system, where they promote cell migration and activation. In the central nervous system, chemokine has been implicated in neuroinflammatory processes. However, an increasing number of evidence suggests that they have regulatory functions in the normal nervous system, where they could participate in cell communication. In this work, using a semiquantitative immunohistochemistry approach, we provide the first neuroanatomical mapping of constitutive neuronal CCR2 localization. Neuronal expression of CCR2 was observed in the anterior olfactory nucleus, cerebral cortex, hippocampal formation, caudate putamen, globus pallidus, supraoptic and paraventricular hypothalamic nuclei, amygdala, substantia nigra, ventral tegmental area, and in the brainstem and cerebellum. These data are largely in accordance with results obtained using quantitative autoradiography with [(125)I]MCP-1/CCL2 and RT-PCR CCR2 mRNA analysis. Furthermore, using dual fluorescent immunohistochemistry we studied the chemical phenotype of labeled neurons and demonstrated the coexistence of CCR2 with classical neurotransmitters. Indeed, localization of CCR2 immunostaining is observed in dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area as well as in cholinergic neurons in the substantia innominata and caudate putamen. Finally, we show that the preferential CCR2 ligand, MCP-1/CCL2, elicits Ca(2+) transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon. In conclusion, the constitutive neuronal CCR2 expression in selective brain structures suggests that this receptor could be involved in neuronal communication and possibly associated with cholinergic and dopaminergic neurotransmission and related disorders.

Aminopeptidase N (APN), which is widely distributed in mammalian tissues, is able to cleave numerous regulatory peptides. The selective inhibitor of APN, [(125)I] RB129, has been used to study the distribution of this exopeptidase during rat prenatal development and adult life by in vitro whole-body autoradiography. In the central nervous system, APN shows a weak labeling compared to the major part of the non-nervous tissues in the embryo and in the adult. APN is progressively expressed in kidney, intestine, heart, lung, sensory organs, eye, and thymus. In organs such as the liver, the cartilages and the bones, altered levels of APN expression are observed during the development, or in the embryo compared to the adult, suggesting a role of APN during the liver haematopoiesis and bone growth. At this time, all the physiological functions of APN are still incompletely known, however its developmental pattern of expression strongly suggests a function of modulation of this enzyme during the development, next in physiological and/or pathological situations in adult. In this way, APN could represent a new therapeutic target in pathological processes, such as tumoral proliferation and/or angiogenesis associated with cancer development, where an increase in the level of this enzyme has been observed.

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein-coupled receptor for the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) was reported to have possible implications in brain development and AIDS-related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF-1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.

To clarify the controversy concerning the role of catecholaminergic systems in regulating hypothalamic corticotropin-releasing factor (CRF) secretion, we assessed the direct effects of norepinephrine and epinephrine, alone and in association with mixed alpha and beta antagonists on hypothalamic CRF secretion. An in vitro rat mediobasal hypothalamus perifusion system was used, in which CRF secretion from a single explant was evaluated by a specific radioimmunoassay. We found that norepinephrine stimulated CRF secretion, with peak effects at 10(-8) M concentration, whereas epinephrine had no effect on CRF secretion. The effect of norepinephrine was antagonised by the mixed alpha antagonist phentolamine and by the mixed beta antagonist propranolol. We conclude that norepinephrine, but not epinephrine, stimulate hypothamic CRF secretion via alpha and beta receptors. The data support the idea that the central noradrenergic systems are excitatory upon CRF-41 secretion when acting directly at the hypothalamic level.

The present work describes time-dependent changes in the content of corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH), and beta-endorphin (beta-EP) in the hypothalamus (HT) and anterior pituitary (AP) and in the concentration of ACTH and beta-EP in the plasma during the 17 beta estradiol (E2) benzoate (E2B)-induced luteinizing hormone (LH) surge in ovariectomized cynomolgus monkeys. Monkeys were euthanized at 0, 30, 48, 72, and 96 hr post-E2B. HT and AP were rapidly dissected, extracted in 2 N acetic acid containing 1 mM phenylmethane sulfonyl fluoride at 4 degrees C, and centrifuged at 18,000g for 30 min. Peptide concentrations were measured in the supernatant by specific radioimmunoassays (RIAs). In the HT, there were significant (P < 0.05) decreases in ACTH and beta-EP content by 30 hr post-E2B and a significant (P < 0.05) decrease in HT CRH content 48 hr post-E2B. Thereafter, CRH, ACTH, and beta-EP content increased up to 72 hr post-E2B. In the AP, there was an almost linear decrease in the CRH content through 48 hr post-E2B followed by a marked 20-fold (P < 0.01) increase in the AP CRH content at 72 hr post-E2B, which corresponds to the time of the descending arm of the LH surge. The patterns of ACTH and beta-EP content were very similar in the AP, while that of CRH differed markedly. In contrast, in the HT CRH, ACTH, and beta-EP profiles were very similar. Significant (P < 0.05) increases in circulating levels of ACTH, beta-EP, and cortisol were evident at 30 hr (all 3 hormones), 48 hr (beta-EP and cortisol), and 72 hr (cortisol) post-E2B, which corresponds with the time of decreased hypothalamic content of CRH, ACTH, and beta-EP. These results suggest that there maybe a marked activation of the hypothalamo-anterior pituitary-adrenal axis during the negative and positive feedback phases of the E2B-induced LH surge in the ovariectomized monkey.