Nociceptive terminals possess the elements for detecting, transmitting, and modulating noxious signals, thus being pivotal for pain sensation. Despite this, a functional description of the transduction process by the terminals, in physiological conditions, has not been fully achieved. Here, we studied how nociceptive terminals in vivo convert noxious stimuli into propagating signals. By monitoring noxiousstimulus-induced Ca 2+ dynamics from mouse corneal terminals, we found that initiation of Na + channel (Nav)-dependent propagating signals takes place away from the terminal and that the starting point for Nav-mediated propagation depends on Nav functional availability. Acute treatment with the proinflammatory cytokines tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b) resulted in a shift of the location of Nav involvement toward the terminal, thus increasing nociceptive excitability. Moreover, a shift of Nav involvement toward the terminal occurs in corneal hyperalgesia resulting from acute photokeratitis. This dynamic change in the location of Nav-mediated propagation initiation could underlie pathological pain hypersensitivity.

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8-expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8 -EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low-threshold, robust responses to cooling. The remaining TRPM8 corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low-firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8 corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low-background activity and abnormal responsiveness to cooling pulses. These morpho-functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age-induced abnormal tearing and to the high incidence of DED in elderly people.

We thank Ardem Patapoutian and Paul Heppenstall for providing the Piezo2 fl/fl and Advillin-Cre mice respectively, and Salvador Sala for helping with current kinetic analysis. Mireille Tora and Eva Quintero are acknowledged for excellent technical assistance and Carlos Ramos and Sergio Javaloy for illustrations. We also thanks all members of the Sensory Transduction and Nociception Group for useful comments and suggestions. The authors gratefully acknowledge the financial support received by the Spanish Government projects: SAF2016-77233R (A.Gomis and F.V.

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca 2ϩ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold-and menthol-evoked intracellular [Ca 2ϩ ] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K ϩ current I KD. Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low-and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(ϩ) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.

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Corneal injuries are one of the most frequently observed ocular diseases, leading to permanent damage and impaired vision if they are not treated properly. In this sense, adequate wound healing after injury is critical for keeping the integrity and structure of the cornea. The goal of this work was to assess the potential of polymeric nanocapsules, either unloaded or loaded with cyclosporine A or vitamin A, alone or in combination with mitomycin C, for the treatment of corneal injuries induced by photorefractive keratectomy surgery. The biopolymers selected for the formation of the nanocapsules were polyarginine and protamine, which are known for their penetration enhancement effect. The results showed that, following topical instillation to a mouse model of corneal injury, all the nanocapsule formulations, either unloaded or loaded with cyclosporine A or vitamin A, were able to stimulate corneal wound healing. In addition, the healing rate observed for the combination of unloaded protamine nanocapsules with mitomycin C was comparable to the one observed for the positive control Cacicol®, a biopolymer known as a corneal wound healing enhancer. Regarding the corneal opacity, the initial grade of corneal haze (>3) induced by the photorefractive keratectomy was more rapidly reduced in the case of the positive control, Cacicol®, than in corneas treated with the nanocapsules. In conclusion, this work shows that drug-free arginine-rich (polyarginine, protamine) nanocapsules exhibit a positive behavior with regard to their potential use for corneal wound healing.

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The cornea is an avascular connective tissue that is crucial, not only as the primary barrier of the eye but also as a proper transparent refractive structure. Corneal transparency is necessary for vision and is the result of several factors, including its highly organized structure, the physiology of its few cellular components, the lack of myelinated nerves (although it is extremely innervated), the tightly controlled hydration state, and the absence of blood and lymphatic vessels in healthy conditions, among others. The avascular, immune-privileged tissue of the cornea is an ideal model to study the interactions between its well-characterized and dense sensory nerves (easily accessible for both focal electrophysiological recording and morphological studies) and the low number of resident immune cell types, distinguished from those cells migrating from blood vessels. This paper presents an overview of the corneal structure and innervation, the resident dendritic cell (DC) subpopulations present in the cornea, their distribution in relation to corneal nerves, and their role in ocular inflammatory diseases. A mouse model in which sensory axons are constitutively labeled with tdTomato and DCs with green fluorescent protein (GFP) allows further analysis of the neuro-immune crosstalk under inflammatory and steady-state conditions of the eye.