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.

Modulation of Inflammation-Related Genes in the Cornea of a Mouse Model of Dry Eye upon Treatment with Cyclosporine Eye Drops.

Purpose/Aim: Inflammation is recognized as playing an etiological role in dry eye disease. This study aimed to assess the efficacy of various topical cyclosporine A (CsA) formulations on cornea inflammatory markers in a mouse model of dry eye. MATERIAL AND METHODS: Six- to 7-week-old mice treated with scopolamine were housed in a controlled environment room to induce dry eye. Following dry eye confirmation by corneal fluorescein staining (CFS), the mice were treated three times a day with: 0.05%CsA (Restasis, Allergan), 0.1%CsA (Ikervis, Santen), 1%CsA oil solution, and 0.5% loteprednol etabonate (LE, Lotemax, Baush+Lomb), or left untreated. Aqueous tear production and CFS scores were assessed during the treatment period, and corneas were collected to measure the expression profile of a selection of inflammatory genes. RESULTS: After 7 days of treatment, the CFS scores were reduced by 21%, 31%, and 44% with 0.05%CsA, 0.1%CsA, and 1%CsA eye drops, respectively. By contrast, 0.5% LE did not decrease corneal fluorescein staining at day 10. A statistically significant dose-dependent CFS reduction was observed only between the 0.05% and 1%CsA formulations. The gene expression profiles indicated that 12, 18, 17 genes were downregulated by 0.05%CsA, 0.1%CsA, 1%CsA, respectively. Among them, the genes significantly downregulated were: IL1A, IL1R1, and TLR4 with 0.05%CsA; H2-Eb1, IL1A, IL1B, IL1RN, IL6, TGFB2, TGFB3, TLR2, TLR3, and TLR4 with 0.1%CsA; IL1B, IL6, TGFB3, and TLR4 with 1%CsA. TGFB1 and TGFBR1 were the only genes upregulated in all groups, but only TGFB1 upregulation reached significance. IL6RA was significantly upregulated by 0.05%CsA. CONCLUSIONS: This study indicates that the three CsA formulations effectively modulated TLR4, TGFß1, IL1, and IL6 pathways to reduce corneal epithelium lesions in a mouse model of severe dry eye. The study also suggests that the different anti-inflammatory eye drops modulated inflammatory genes in a slightly different manner.

Intraocular pressure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma.

INTRODUCTION: Glaucoma is a sight-threatening retinal neuropathy associated with elevated intraocular pressure (IOP) due to degeneration and fibrosis of the trabecular meshwork (TM). Glaucoma medications aim to reduce IOP without targeting the specific TM pathology, Bone-marrow mesenchymal stem cells (MSCs) are used today in various clinical studies. Here, we investigated the potential of MSCs therapy in an glaucoma-like ocular hypertension (OHT) model and decipher in vitro the effects of MSCs on primary human trabecular meshwork cells. METHODS: Ocular hypertension model was performed by cauterization of 3 episcleral veins (EVC) of Long-Evans male rat eyes. MSCs were isolated from rat bone marrow, amplified in vitro and tagged with quantum dot nanocrystals. Animals were distributed as 1) MSCs group receiving 5.10(5)cells/6µl Minimum Essential Medium and 2) MEM group receiving 6µl MEM (n = 10 each). Injections were performed into the anterior chamber of 20 days-hypertensive eyes and IOP was monitored twice a week for 4 weeks. At the end of experiment, cell distribution in the anterior segment was examined in confocal microscopy on flat mounted corneas. Moreover, we tested in vitro effects of MSCs conditioned medium (MSC-CM) on primary human trabecular meshwork cells (hTM cells) using Akt activation, myosin phosphorylation and TGF-ß2-dependent profibrotic phenotype in hTM cells. RESULTS: We demonstrated a rapid and long-lasting in vivo effect of MSCs transplantation that significantly reduced IOP in hypertensive eyes induced by EVC. MSCs were located to the ciliary processes and the TM. Enumeration of RGCs on whole flat-mounted retina highlighted a protective effect of MSCs on RGCs death. In vitro, MSC-CM promotes: (i) hTM cells survival by activating the antiapoptotic pathway, Akt, (ii) hTM cells relaxation as analyzed by the decrease in myosin phosphorylation and (iii) inhibition of TGF-ß2-dependent profibrotic phenotype acquisition in hTM cells. CONCLUSIONS: MSCs injection in the ocular anterior chamber in a rat model of OHT provides neuroprotective effect in the glaucoma pathophysiology via TM protection. These results demonstrate that MSCs constitute promising tool for treating ocular hypertension and retinal cell degeneration.

Cellular and subcellular evidence for neuronal interaction between the chemokine stromal cell-derived factor-1/CXCL 12 and vasopressin: regulation in the hypothalamo-neurohypophysial system of the Brattleboro rats.

We previously described a colocalization between arginine vasopressin (AVP) and the chemokine stromal cell-derived factor-1alpha (SDF-1) in the magnocellular neurons of both the hypothalamic supraoptic and paraventricular nucleus as well as the posterior pituitary. SDF-1 physiologically affects the electrophysiological properties of AVP neurons and consequently AVP release. In the present study, we confirm by confocal and electron microscopy that AVP and SDF-1 have a similar cellular distribution inside the neuronal cell and can be found in dense core vesicles in the nerve terminals in the posterior pituitary. Because the Brattleboro rats represent a good model of AVP deficiency, we tested in these animals the fate of SDF-1 and its receptor CXCR4. We identified by immunohistochemistry that both SDF-1 and CXCR4 immunoreactivity were strongly decreased in Brattleboro rats and were strictly correlated with the expression of AVP protein in supraoptic nucleus, paraventricular nucleus, and the posterior pituitary. We observed by real-time PCR an increase in SDF-1 mRNA in both heterozygous and homozygous rats. The effect on the SDF-1/CXCR4 system was not linked to peripheral modifications of kidney water balance because it could not be restored by chronic infusion of deamino-8D-ariginine-vasopressin, an AVP V2-receptor agonist. These original data further suggest that SDF-1 may play an essential role in the regulation of water balance.

Chemokines: a new class of neuromodulator?

Chemokines are not only found in the immune system or expressed in inflammatory conditions: they are constitutively present in the brain in both glial cells and neurons. Recently, the possibility has been raised that they might act as neurotransmitters or neuromodulators. Although the evidence is incomplete, emerging data show that chemokines have several of the characteristics that define neurotransmitters. Moreover, their physiological actions resemble those of neuromodulators in the sense that chemokines usually have few effects by themselves in basal conditions, but modify the induced release of neurotransmitters or neuropeptides. These findings, together with the pharmacological development of agonists and antagonists that are selective for chemokine receptors and can cross the blood-brain barrier, open a new era of research in neuroscience.

Chemokines and chemokine receptors in the brain: implication in neuroendocrine regulation.

Chemokines are small secreted proteins that chemoattract and activate immune and non-immune cells both in vivo and in vitro. In addition to their well-established role in the immune system, several recent reports have suggested that chemokines and their receptors may also play a role in the central nervous system (CNS). The best known central action is their ability to act as immunoinflammatory mediators. Indeed, these proteins regulate leukocyte infiltration in the brain during inflammatory and infectious diseases. However, we and others recently demonstrated that 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. The goal of this review is to highlight the role of chemokines in the control of neuroendocrine functions. First, we will focus on the expression of chemokines and their receptors in the CNS, with the main spotlight on the neuronal expression in the hypothalamo-pituitary system. Secondly, we will discuss the role--we can now suspect--of chemokines and their receptors in the regulation of neuroendocrine functions. In conclusion, we propose that chemokines can be added to the well-described neuroendocrine regulatory mechanisms, providing an additional fine modulatory tuning system in physiological conditions.

Chemokines and brain functions.

Chemokines are small secreted proteins that chemoattract and activate immune and non-immune cells both in vivo and in vitro. Besides their well-established role in the immune system, several recent reports have suggested that chemokines and their receptors may also play a role in the central nervous system (CNS). The best-known central action is their ability to act as immuno-inflammatory mediators. Indeed, these proteins regulate the leukocyte infiltration in the brain during inflammatory and infectious diseases. However, recent studies clearly demonstrate that chemokines and their receptors are constitutively expressed by glial and neuronal cells in the CNS, where they are involved in intercellular communication. The goal of this review is to summarize recent information concerning the role of chemokines in brain functions. The first part will focus on the expression of chemokines and their receptors in the CNS with the main spotlight on the neuronal expression. In the second part, we will discuss the role of chemokines and their receptors in normal brain physiology. Because several chemokines are involved in neuroinflammatory and neurodegenerative disorders, the role of chemokines and their receptors in these diseases is reviewed further in this section. In conclusion, the implication of chemokines in cellular communication could allow: i) to identify a new pathway for neuron-neuron and/or glia-glia and/or neuron-glia communications that are relevant to both normal brain function and neuroinflammatory and neurodegenerative diseases; ii) to develop new therapeutic approaches for still untreatable diseases further.

Highly regionalized neuronal expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) in rat brain: evidence for its colocalization with neurotransmitters and neuropeptides.

The monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 are key modulators of immune functions. In the nervous system, MCP-1/CCL2 is implicated in neuroinflammatory pathologies. However, cerebral functions of MCP-1/CCL2 under normal conditions are still unclear. In this study, using reverse transcriptase-polymerase chain reaction (RT-PCR) and specific rat MCP-1 enzyme-linked immunosorbent assay (ELISA) approaches, we observed that MCP-1/CCL2 mRNA and protein were expressed in different punched regions of the normal rat central nervous system. Immunohistochemical studies further revealed that this chemokine is constitutively expressed not only in astrocytes but also in neurons, in discrete neuroanatomical regions. Neuronal expression of MCP-1/CCL2 is mainly found in the cerebral cortex, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, facial nuclei, motor and spinal trigeminal nuclei, and gigantocellular reticular nucleus and in Purkinje cells in the cerebellum. Moreover, we obtained the first evidence that MCP-1/CCL2 is constitutively expressed in cholinergic neurons, notably in the magnocellular preoptic and oculomotor nuclei, and in dopaminergic neurons of the substantia nigra pars compacta. In addition, in the lateral hypothalamic area, MCP-1/CCL2 co-localized with melanin-concentrating hormone-expressing neurons. Interestingly, we demonstrate a co-localization of MCP-1/CCL2 with vasopressin in magnocellular neuronal cell bodies and processes in the supraoptic and paraventricular hypothalamic nuclei, as well as in processes in the internal layer of the median eminence and in the posterior pituitary. Taken together, our data suggest that MCP-1/CCL2 could act as a modulator of neuronal activity and neuroendocrine functions.

Highly regionalized distribution of stromal cell-derived factor-1/CXCL12 in adult rat brain: constitutive expression in cholinergic, dopaminergic and vasopressinergic neurons.

The stromal cell-derived factor-1 (SDF-1)/CXCL12 and its receptor CXCR4 are key modulators of immune functions. In the nervous system, SDF-1/CXCL12 is crucial for neuronal guidance in developing brain, intercellular communication and the neuropathogenesis of acquired immunodeficiency syndrome. However, cerebral functions of SDF-1/CXCL12 in adult brain are poorly understood. The understanding of its role in the adult brain needs a detailed neuroanatomical mapping of SDF-1/CXCL12. By dual immunohistochemistry we demonstrate that this chemokine is constitutively expressed not only in astrocytes and microglia but also in neurons, in discrete neuroanatomical regions. Indeed, neuronal expression of SDF-1/CXCL12 is mainly found in cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provide the first evidence that SDF-1/CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. Interestingly we also show, for the first time, a selective co-localization of SDF-1/CXCL12 with vasopressin-expressing neurons in the supraoptic and paraventricular hypothalamic nuclei. In addition, in the lateral hypothalamic area, SDF-1/CXCL12 was found to be located on melanin concentrating hormone-expressing neurons. Altogether, these original data suggest that SDF-1/CXCL12 could be a modulatory neuropeptide regulating both central cholinergic and dopaminergic systems. In addition, a key role for SDF-1/CXCL12 in neuroendocrine regulation of vasopressin-expressing neurons represents an exciting new field of research.