Evaluation of neuroprotective and immunomodulatory properties of mesenchymal stem cells in an ex vivo retinal explant model.

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, IL1ß, 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.

An overview of current alternative models in the context of ocular surface toxicity.

The 21st century has seen a steadily increasing social awareness of animal suffering, with increased attention to ethical considerations. Developing new integrated approaches to testing and assessment (IATA) strategies is an Organisation for Economic Co-operation and Development (OECD) goal to reduce animal testing. Currently, there is a lack of alternative models to test for ocular surface toxicity (aside from irritation) in lieu of the Draize eye irritation test (OECD guideline No. 405) performed in rabbits. Five alternative in vitro or ex vivo methods have been validated to replace this reference test, but only in combination. However, pathologies like Toxicity-Induced Dry Eye (TIDE), cataract, glaucoma, and neuropathic pain can occur after exposure to a pharmaceutical product or chemical and therefore need to be anticipated. To do so, new models of lacrimal glands, lens, and neurons innervating epithelia are required. These models must take into account real-life exposure (dose, time, and tear film clearance). The scientific community is working hard to develop new, robust, alternative, in silico, and in vitro models, while attempting to balance ethics and availability of biological materials. This review provides a broad overview of the validated methods for analyzing ocular irritation and those still used by some industries, as well as promising models that need to be optimized according to the OECD. Finally, we give an overview of recently developed innovative models, which could become new tools in the evaluation of ocular surface toxicity within the scope of IATAs.

Association of Sensitive Eyes with Sensitive Skin: A Worldwide Study of 10,743 Subjects.

INTRODUCTION: Sensitive eyes are commonly reported by patients, but there are very few epidemiological studies on this disorder. The aim of this study was the evaluation of the self-reported frequency of sensitive eyes and the association with sensitive skin. METHODS: A survey was performed on a representative sample of the population aged more than 18 years in five different countries (Brazil, China, France, Russia, and the USA). All participants answered a questionnaire on sociodemographic characteristics; skin phototype; eye color; tobacco consumption; exposure to sunlight, air pollution, or having pets; and sleep disorders. The presence of sensitive eyes, eyelids, or skin and their triggering factors were assessed with specific questions. RESULTS: A total of 10,743 individuals (5,285 men and 5,458 women) were included in the study. Among them, 48.2% reported having sensitive skin and 46.0% reported having sensitive eyes. Sensitive eyes were more frequently reported by women (46.5%) than men (39.4%) in all countries, with the exception of China. The presence of sensitive eyes was more frequent if skin was very sensitive. More than half of subjects with sensitive eyes declared that their triggering factors were exposure to sunlight, dust, touch pad screens, or computer screens or dry air. They were more exposed to pollution and tobacco. Their phototype (including eye color) was lighter. DISCUSSION/CONCLUSION: This large study shows that self-declared sensitive eyes are very frequent and commonly associated with sensitive skin. Triggering factors of sensitive eyes are more specific.

Shhedding New Light on the Role of Hedgehog Signaling in Corneal Wound Healing.

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.

Electrical match between initial segment and somatodendritic compartment for action potential backpropagation in retinal ganglion cells.

The action potential of most vertebrate neurons initiates in the axon initial segment (AIS) and is then transmitted to the soma where it is regenerated by somatodendritic sodium channels. For successful transmission, the AIS must produce a strong axial current, so as to depolarize the soma to the threshold for somatic regeneration. Theoretically, this axial current depends on AIS geometry and Na+ conductance density. We measured the axial current of mouse retinal ganglion cells using whole cell recordings with post hoc AIS labeling. We found that this current is large, implying high Na+ conductance density, and carries a charge that covaries with capacitance so as to depolarize the soma by ∼30 mV. Additionally, we observed that the axial current attenuates strongly with depolarization, consistent with sodium channel inactivation, but temporally broadens so as to preserve the transmitted charge. Thus, the AIS appears to be organized so as to reliably backpropagate the axonal action potential.NEW & NOTEWORTHY We measured the axial current produced at spike initiation by the axon initial segment of mouse retinal ganglion cells. We found that it is a large current, requiring high sodium channel conductance density, which covaries with cell capacitance so as to ensure a ∼30 mV depolarization. During sustained depolarization the current attenuated, but it broadened to preserve somatic depolarization. Thus, properties of the initial segment are adjusted to ensure backpropagation of the axonal action potential.

Capsazepine decreases corneal pain syndrome in severe dry eye disease.

BACKGROUND: Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. METHODS: Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. RESULTS: First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. CONCLUSION: These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

Opioid and chemokine receptor crosstalk: a promising target for pain therapy?

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.

Dialing in the dialogue between inflammation and the brain.

The nervous and immune systems communicate with one another and jointly influence functional responses. To highlight the many advances on this hot topic, Brain, Behavior, and Immunity conceptualized a Special Issue entitled "Dialing in the Dialogue Between Inflammation and the Brain." Recent advances and exciting developments in understanding communication pathways between the brain and the immune system during both physiological and pathological insults are highlighted.

TRPM8: A Therapeutic Target for Neuroinflammatory Symptoms Induced by Severe Dry Eye Disease.

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.

Chronic dry eye induced corneal hypersensitivity, neuroinflammatory responses, and synaptic plasticity in the mouse trigeminal brainstem.

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-1ß), 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-1ß, 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.

Correction to: Tyrosine-hydroxylase immunoreactivity in the mouse transparent brain and adrenal glands.

Unfortunately, the given name and family name of the fourth author was incorrectly tagged in the xml data, therefore it is abbreviated wrongly as ''Goazigo AR'' in Pubmed. The correct given name is Annabelle and family name is Reaux­Le Goazigo.

Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Angiogenesis is a cause of visual impairment and blindness in the wet form of age-related macular degeneration and in ischemic retinopathies. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization (CNV) and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF and possible adverse effects of long-term VEGF inhibition in the retina and choroid highlight a need for additional alternative therapies. Integrins αvß3 and αvß5, which regulate endothelial cell proliferation and stabilization, have been implicated in ocular angiogenesis. Lebecetin (LCT) is a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom and interacts with α5ß1- and αv-containing integrins. We previously showed that LCT inhibits human brain microvascular endothelial cell adhesion, migration, proliferation, and tubulogenesis. To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on CNV in the mouse CNV model and on retinal neovascularization in the oxygen-induced retinopathy model. Our data demonstrate that a single injection of LCT efficiently reduced CNV and retinal neovascularization in these models.-Montassar, F., Darche, M., Blaizot, A., Augustin, S., Conart, J.-B., Millet, A., Elayeb, M., Sahel, J.-A., Réaux-Le Goazigo, A., Sennlaub, F., Marrakchi, N., Messadi, E., Guillonneau, X. Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Proinflammatory Markers, Chemokines, and Enkephalin in Patients Suffering from Dry Eye Disease.

Dry eye symptoms are among the leading complaints in ophthalmology. Dry eye disease (DED) is associated with significant pain affecting quality of life. Cellular and molecular mechanisms underlying ocular pain associated with DED are not fully understood. In this study, we investigated the ocular surface of patients with DED using in vivo confocal microscopy (IVCM) to quantify corneal nerve density and its relation with corneal inflammation. Gene expression of the proinflammatory markers HLA-DR, IL-6, CXCL12, and CCL2 and the receptors CXCR4 and CCR2, as well as PENK (enkephalin precursor), was therefore quantified in conjunctival impression cytology specimens. Thirty-two patients with DED and 15 age-matched controls were included. Subbasal nerve density was significantly lower in DED patients compared to controls. IVCM analysis revealed that DED patients had a significantly higher corneal dendritic cell density compared to controls. Conjunctival impression cytology analysis revealed that HLA-DR, IL-6, CXCR4, and CCL2/CCR2 mRNA levels were significantly increased in DED patients compared to controls, whereas PENK mRNA levels were significantly decreased. Similar results were obtained in vitro on immortalized human conjunctiva-derived epithelial cells challenged with osmotic stress that mimics the DED condition. These results demonstrate that proinflammatory molecules and endogenous enkephalin have opposite gene regulation during DED.

Ocular inflammation induces trigeminal pain, peripheral and central neuroinflammatory mechanisms.

Ocular surface diseases are among the most frequent ocular pathologies, with prevalence ranging from 20% of the general population. In addition, ocular pain following corneal injury is frequently observed in clinic. The aim of the study was to characterize the peripheral and central neuroinflammatory process in the trigeminal pathways in response to cornea alteration induced by chronic topical instillations of 0.2% benzalkonium chloride (BAC) in male C57BL/6J mice. In vitro BAC induced neurotoxicity and increases neuronal (FOS, ATF3) and pro-inflammatory (IL-6) markers in primary mouse trigeminal ganglion culture. BAC-treated mice exhibited 7days after the treatment reduced aqueous tear production and increased inflammatory cell infiltration in the cornea. Hypertonic saline-evoked eye wipe behavior was enhanced in BAC-treated animals that exhibited increased FOS, ATF3 and Iba1 immunoreactivity in the trigeminal ganglion. Ocular inflammation is associated with a significant increase in IL-6 and TNF-α mRNA expression in the trigeminal ganglion. We reported a strong increase in FOS and Iba1 positive cells in particular in the sensory trigeminal complex at the ipsilateral interpolaris/caudalis (Vi/Vc) transition and Vc/upper cervical cord (Vc/C1) regions. In addition, activated microglial cells were tightly wrapped around activated FOS neurons in both regions and phosphorylated p38 mitogen-activated protein kinase was markedly enhanced specifically in microglial cells during ocular inflammation. Similar data were obtained in the facial motor nucleus. These neuroanatomical data correlated with the increase in mRNA expression of pro-inflammatory (TNF-α, IL-6, CCL2) and neuronal (FOS and ATF3) markers. Interestingly, the suppression of corneal inflammation 10days following the end of BAC treatment resulted in a marked attenuation of peripheral and central changes observed in pathological conditions. This study provides the first demonstration that corneal inflammation induces activation of neurons and microglial p38 MAPK pathway within sensory trigeminal complex. These results suggest that this altered activity in intracellular signaling caused by ocular inflammation might play a priming role in the central sensitization of ocular related brainstem circuits, which represents a significant factor in ocular pain development.

Bilateral neuroinflammatory processes in visual pathways induced by unilateral ocular hypertension in the rat.

BACKGROUND: Glaucoma is one of the leading causes of irreversible blindness in the world. The major risk factor is elevated intraocular pressure (IOP) leading to progressive retinal ganglion cell (RGC) death from the optic nerve (ON) to visual pathways in the brain. Glaucoma has been reported to share mechanisms with neurodegenerative disorders. We therefore hypothesize that neuroinflammatory mechanisms in central visual pathways may contribute to the spread of glaucoma disease. The aim of the present study was to analyze the neuroinflammation processes that occur from the pathological retina to the superior colliculi (SCs) in a rat model of unilateral ocular hypertension induced by episcleral vein cauterization (EVC). RESULTS: Six weeks after unilateral (right eye) EVC in male Long-Evans rats, we evaluated both the neurodegenerative process and the neuroinflammatory state in visual pathway tissues. RGCs immunolabeled (Brn3a(+)) in ipsilateral whole flat-mounted retina demonstrated peripheral RGC loss associated with tissue macrophage/microglia activation (CD68(+)). Gene expression analysis of hypertensive and normotensive retinas revealed a significant increase of pro-inflammatory genes such as CCL2, IL-1ß, and Nox2 mRNA expression compared to naïve eyes. Importantly, we found an upregulation of pro-inflammatory markers such as IL-1ß and TNFα and astrocyte and tissue macrophage/microglia activation in hypertensive and normotensive RGC projection sites in the SCs compared to a naïve SC. To understand how neuroinflammation in the hypertensive retina is sufficient to damage both right and left SCs and the normotensive retina, we used an inflammatory model consisting in an unilateral stereotaxic injection of TNFα (25 ng/µl) in the right SC of naïve rats. Two weeks after TNFα injection, using an optomotor test, we observed that rats had visual deficiency in both eyes. Furthermore, both SCs showed an upregulation of genes and proteins for astrocytes, microglia, and pro-inflammatory cytokines, notably IL-1ß. In addition, both retinas exhibited a significant increase of inflammatory markers compared to a naïve retina. CONCLUSIONS: All these data evidence the complex role played by the SCs in the propagation of neuroinflammatory events induced by unilateral ocular hypertension and provide a new insight into the spread of neurodegenerative diseases such as glaucoma.

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion.

Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 µm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

Stromal cell-derived CCL2 drives neuropathic pain states through myeloid cell infiltration in injured nerve.

Neuropathic pain resulting from peripheral nerve injury involves many persistent neuroinflammatory processes including inflammatory chemokines that control leukocyte trafficking and activate resident cells. Several studies have shown that CCL2 chemokine, a potent attractant of monocytes, and its cognate receptor, CCR2, play a critical role in regulating nociceptive processes during neuropathic pain. However, the role of CCL2 in peripheral leukocyte infiltration-associated neuropathic pain remains poorly understood. In particular, the contribution of individual CCL2-expressing cell populations (i.e. stromal and leukocytes) to immune cell recruitment into the injured nerve has not been established. Here, in preclinical model of peripheral neuropathic pain (i.e. chronic constriction injury of the sciatic nerve), we have demonstrated that, CCL2 content was increased specifically in nerve fibers. This upregulation of CCL2 correlated with local monocyte/macrophage infiltration and pain processing. Furthermore, sciatic intraneural microinjection of CCL2 in naïve animals triggered long-lasting pain behavior associated with local monocyte/macrophage recruitment. Using a specific CCR2 antagonist and mice with a CCL2 genetic deletion, we have also established that the CCL2/CCR2 axis drives monocyte/macrophage infiltration and pain hypersensitivity in the CCI model. Finally, specific deletion of CCL2 in stromal or immune cells respectively using irradiated bone marrow-chimeric CCI mice demonstrated that stromal cell-derived CCL2 (in contrast to CCL2 immune cell-derived) tightly controls monocyte/macrophage recruitment into the lesion and plays a major role in the development of neuropathic pain. These findings demonstrate that in chronic pain states, CCL2 expressed by sciatic nerve cells predominantly drove local neuro-immune interactions and pain-related behavior through CCR2 signaling.

Src family kinases involved in CXCL12-induced loss of acute morphine analgesia.

Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occurred in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu-(MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1µg) significantly attenuated the subcutaneous morphine (4mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5µg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10µg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12-induced loss of acute morphine analgesia is linked to Src family kinases activation.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury.

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.