A new R,R-RvD6 isomer with protective actions following corneal nerve injury

Abstract

The transparent cornea is the most densely innervated tissue in the body, primarily by sensory nerves originating from the trigeminal ganglia (TG). Damage to corneal nerves reduces sensitivity and tear secretion and results in dry eye. Consequently, ocular pain, for which no satisfactory therapies exist, arises in many cases. Treatment of injured corneas with pigment epithelium-derived factor (PEDF) combined with docosahexaenoic acid (DHA) stimulates nerve regeneration in models of refractive surgery, which damages nerves. The mechanism involves the synthesis of a stereoisomer of resolvin D6 (R,R-RvD6) formed after incorporating DHA into membrane lipids. Activation of a PEDF receptor (PEDF-R) with phospholipase activity releases DHA to synthesize the new resolvin isomer, which is secreted via tears. Topical treatment of mice corneas with R,R-RvD6 shows higher bioactivity in regenerating nerves and increasing sensitivity compared to PEDF+DHA. It also stimulates a transcriptome in the TG that modulates genes involved in ocular pain. Our studies suggest an important therapeutic role for R,R-RvD6 in regenerating corneal nerves and decreasing pain resulting from dry eye.

1. Introduction

The cornea is an avascular, transparent tissue located at the front of the eye, enabling light to pass through the lens and reach the retina. It stands as the eye’s most potent refractive component and consists of three primary layers. The outermost layer, known as the epithelium, makes direct contact with tears and comprises 6–7 layers of cells [1]. The basal cells within this layer are highly proliferative, facilitating rapid coverage of corneal wounds. The thickest layer, called the stroma, is composed of collagen fibers arranged in a manner that imparts transparency to the tissue [2]. Scattered among these fibers are keratocytes, largely quiescent cells responsible for maintaining collagen and other extracellular matrix (ECM) components within the stroma. This layer serves to safeguard the eye and uphold the integrity of the visual axis. The innermost layer, known as the endothelium, consists of a monolayer of cells featuring specialized pumps that prevent stromal swelling [3]. In humans, endothelial cells exhibit limited mitosis and tend to elongate with age, covering the posterior portion of the cornea.

One notable characteristic of the cornea is its dense innervation, predominantly by sensory nerves stemming from the ophthalmic branch of the trigeminal ganglia (TG). In 2010, we showed the complete nerve architecture of the human cornea for the first time [4]. Through consecutive images of corneal nerves stained with an anti-β III tubulin antibody, we captured the tissue’s intricate innervation pattern, spanning from the center to the periphery and from the anterior to the posterior regions [5]–[9]. Employing a fluorescent microscope in a time-lapse mode, these images unveiled the intense innervation of the tissue, with abundant free nerve terminals within the epithelium. These terminals contain receptors responsive to touch, pain, and temperature sensations [10], rendering the cornea significantly more sensitive—approximately 300 to 500 times more than the skin.

Moreover, several neuropeptides have been identified within the corneal nerve network. These include Substance P (SP) [3], calcitonin gene-related peptide (CGRP) [4], [11], and galanin (GAL) [12] of sensory origin; catecholamines [13] and neuropeptide Y (NPY) [12] of sympathetic origin; and GAL [12], cholecystokinin (CCK) [14], vasoactive intestinal peptide (VIP) [12], and methionine-enkephalin (M-ENK) [12] of parasympathetic origin. Notably, SP and CGRP are particularly significant, as they are the major contributors to overall corneal innervation[4], [7]–[9], [15].

In this review, we focused on the therapeutic application of DHA-derived lipid mediators, especially the new isomer R,R-RvD6 in regenerating corneal nerves to sustain the corneal nerve and homeostasis.

2. Importance of corneal nerves to maintain tissue homeostasis

The robust innervation of the cornea plays a pivotal role in maintaining the homeostatic balance of the ocular surface. Consequently, any damage inflicted upon corneal nerves precipitates a reduction in tear production and blinking reflex, alongside compromised epithelial wound healing. These changes, in turn, lead to transparency loss and impaired vision [16], [17]. Several conditions can disrupt corneal innervation, including aging [4], diabetes [18], [19], herpes simplex virus and other infections [5], [20], [21], Sjogren’s syndrome, chemical burns [6], and prolonged use of contact lenses. Of particular importance are refractive surgeries like laser in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and small incision lenticule extraction (SMILE), all techniques that cause damage to the corneal nerves [22]–[24]. While the majority of these procedures are safe, the sheer volume of surgeries performed annually (ranging from 600,000 to 700,000 surgeries per year in the USA alone) has led to an escalation in the number of patients facing nerve-related issues (3) [25]. Such corneal nerve damage can lead to reduced corneal sensitivity, subsequently triggering dry eye disease, which, in severe cases, can induce neuropathic pain, ulcers, and the need for corneal transplants [22]. Recovery of corneal nerves after LASIK can span 3 to 15 years [26], [27].

The ocular surface’s lubrication is ensured by the lacrimal function unit, which encompasses the lacrimal gland, cornea, conjunctiva, meibomian gland, lids, and the sensory and motor nerves that interconnect them [28]. Afferent neurons innervating the cornea respond to surface damage by increasing lacrimation, blinking, and evoking sensations of irritation and pain [29]. The loss of afferent sensory input to the lacrimal gland results in reduced lacrimal secretion, diminished nutritional support, and, as a consequence, a dry ocular surface. Affecting 8%−15% of adults in the USA, dry eye exhibits an increased prevalence with age [25].

3. Corneal nerve regeneration needs the combination of PEDF+DHA

Several clinical studies have explored the potential benefits of omega-3 fatty acids in addressing dry eye conditions. Among these, the Women’s Health Study stands out due to its extensive recruitment of 32,470 female participants. This study revealed a correlation between higher intake of omega-3 fatty acids and reduced prevalence of dry eye syndrome [30]. However, other studies that involved supplementing patients with dry eye using omega-3 fatty acids for 12 months, as compared to a placebo, did not find differences in dry eye symptoms [27,28].

Previous investigations conducted in our laboratory demonstrated that the administration of pigment epithelium-derived factor (PEDF), a neurotrophic protein, along with docosahexaenoic acid (DHA), facilitates nerve regeneration in rabbit and mouse models of refractive surgery, diabetes, and herpes simplex virus infections [15,29,30].

To assess whether treatment with PEDF or DHA alone post-corneal surgery enhances nerve regeneration, we performed a lamellar keratectomy in rabbits [29]. This procedure, commonly employed in corneal transplants, involves dissecting the corneal nerves that converge radially in the stroma, resulting in substantial nerve damage. After treatment with PEDF+DHA, the percentage of β-tubulin staining increased significantly (Fig. 1) [31]. When treatments were applied separately, no significant differences in nerve density were observed, suggesting that PEDF potentially activates the synthesis of docosanoids derived from DHA and that DHA needs to be added since previous studies had indicated that DHA is present in very low concentrations in the cornea [32].

Fig. 1. Increase in nerve density in rabbits after experimental surgery and different treatments.

New Zealand white rabbits underwent lamellar Keratectomy surgery and then topically treated with vehicle, DHA, PEDF, and PEDF+DHA for 6 weeks. The percentage of nerve density was tested at 8 weeks after surgery and treatments. Control was non-injured rabbits. Significant differences with respect to vehicle only with PEDF+DHA treatment, ANOVA test. Modified from Cortina et al. (Reference 31 in this review).

The data further suggests that PEDF exerts its influence by stimulating a PEDF-receptor (PEDF-R), which possesses phospholipase activity. A PEDF-R originating from a patatin-like phospholipase A2 gene and known as calcium-independent phospholipase A2ζ or adipose triglyceride lipase (ATLG) has been identified. The receptor’s structure has been elucidated by Patricia Becerra’s laboratory [33] and consists of a four-transmembrane protein featuring two extracellular loops and three intracellular domains, with the active site of a Ca2+-independent PLA2 (iPLA2ζ) situated between the second and third transmembrane domain [32]. Considering the scarce presence of DHA in corneal membrane lipids, supplementing with DHA becomes crucial to stimulate corneal nerve regeneration.

Through mass spectrometry analysis, we investigate the incorporation of DHA into the major phospholipids in corneal membranes—phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species. Injured mice corneas were topically treated with DHA or vehicle for 1 hour at 30-minute intervals. It was observed that molecular species containing DHA were notably lower in proportion than those containing arachidonic acid (AA) in the vehicle-treated corneas. This proportion shifted after a 1-hour DHA treatment [34]. PE species showed a greater incorporation of DHA compared to PC. The most pronounced increase was noted in PC and PE species, where DHA was esterified in both sn-1 and sn-2 glycerol positions. While treatment with PEDF alone (excluding DHA) did not alter the levels of these species, treatment with PEDF in the presence of DHA resulted in a significant reduction of PC-22:6/22:6 and PE-22:6/22:6 species. This indicates that PEDF releases DHA from PC and PE species upon activating its receptor with phospholipase A2 activity [34].

4. Synthesis of a new bioactive resolvin D6 stereoisomer after PEDF+DHA stimulation

We also hypothesize that introducing DHA in the presence of PEDF will shift the lipid response in the cornea from eicosanoids derived from AA to docosanoids. To investigate this hypothesis, we treated injured corneas with PEDF+DHA and collected tears for analysis through LC-MS/MS. A distinct peak emerged in the stimulated tears, just preceding the internal standard LTB4-d4. This peak exhibited a parent mass corresponding to a dihydroxy-DHA [35]. The peak shared a comparable fragmentation pattern and UV spectrum with resolvin D6 (RvD6). Resolvins (RvD1-RvD6) are a family of lipid mediators derived from DHA, recognized for their potent bioactive properties in reducing inflammation and promoting resolution [36]–[38]. However, the peak has a shorter retention time than pure RvD6, suggesting that a stereoisomer of the docosanoid is released in the tears.

RvD6 possesses hydroxy groups at carbons (C)17 and C4. The synthesis of 17(S)-hydroxy at C17 of DHA is initiated by a 15-lipoxygenase, followed by the formation of the 4S,17S RvD6 through a 5-lipoxygenase [35]. Alternatively, hydrolysis can confer an R configuration at the C4 position. DHA can also undergo catalysis by the P450 cytochrome system, leading to the formation of 17R-monohydroxy-DHA, followed by hydrolysis yielding 4R, 17R-RvD6. Consequently, four possible isomers of RvD6 exist. When organ culture injured corneas were incubated with inhibitors targeting enzymes capable of synthesizing distinct RvD6 stereoisomers and then stimulated with PEDF+DHA, only Fluvoxamine, an inhibitor of the cytochrome P450 system, significantly reduced the peak’s synthesis. This suggests that the RvD6 isomer could be a 4R-17R dihydroxy-DHA or a 4S-17R dihydroxy-DHA [35].

To investigate the structure of the isomer, we undertook the pure chemical synthesis of R,R and S,R-RvD6 [39]. The RvD6 derived from mouse tears matched the retention time of pure R,R-RvD6 [35]. Upon comparing the biological activity of chemically synthesized pure R,R-RvD6 [34] with PEDF+DHA treatment in mice with injured corneas, it was observed that only the R,R-RvD6 treatment led to a significant increase in cornea sensitivity at day 3 post-injury while other treated condition showed the significant recovery at later investigated time point (day 6 and day 9). Moreover, the nerve density measured at day 12 post-injury demonstrated a higher percentage of corneal nerve density following R,R-RvD6 treatment (Fig. 2A). This effect was also found in S,R- RvD6 and PEDF+DHA treatment with the lower efficacy. The suggested biological synthetic pathway was shown in Figure 2B.

Fig. 2. Percent of recovery of subbasal corneal nerves and sensitivity.

(A) Mouse injured corneas were topically treated 3xday with vehicle, PEDF+DHA or R,R-RvD6. Sensitivity was measured with a Belmonte non-contact corneal esthesiometer three days after injury and treatment. Corneal nerve density was measured in whole mount images of subbasal nerves stained with monoclonal anti-PGP9.5 antibody twelve days after injury. Percent of recovery respect to normal corneas, p values are calculated in comparison to vehicle-treated mice by one-way ANOVA. Modified from Pham TL et al. (Reference 40 in this review). (B) The suggested biological synthetic pathway of RvD6 and its isomers. The pathway of R,R-RvD6 was highlighted in the red oval. Modified from Pham TL et al. (Reference 40 in this review).

5. R,R-RvD6 modulates genes involved in DELP

During refractive surgeries like LASIK, epithelial and stromal nerves are cut to create a flap, and further damage is inflicted through laser photoablation. In certain susceptible patients, this could lead to inflammation and persistent post-operative dry eye symptoms, culminating in a condition known as dry eye-like pain (DELP) characterized by altered corneal innervation. However, the mechanisms behind DELP remain largely unexplored [40]. Symptoms of DELP are a burning sensation, photophobia, and intense eye pain [41]–[45].

Previous studies have indicated that the TG contains receptors and signal molecules that exert influence over the development and persistence of neuropathic pain [43]. The specificity of ocular sensory receptors arises from the expression of transient receptor potential (TRP) channels, a family of calcium and sodium channels that depolarize cells. Various types of sensory nerves are activated depending on the stimulus applied to the cornea (mechanical, polymodal, and cold), resulting in differing degrees of irritation and pain sensation [43,44]. Reduced expression of transient receptor potential melastatin 8 (TRPM8) has been shown to contribute to allodynia and neuropathic pain [45–49]. In a mouse model of a refractive wound, we observed that TRPM8-positive terminals reached only 50% of their normal level after 3 months of injury, suggesting inadequate recovery might contribute to post-surgery DELP sensations [46].

To ascertain the transcriptional mechanism stimulated by R,R-RvD6 in the trigeminal ganglia, we treated injured mouse corneas with vehicle, RvD6 (S,S-RvD6), or our novel lipid, R,R-RvD6, three times a day. We collected TG at 12 days after treatment and subjected them to RNA sequencing [47]. The Venn diagram showed that out of the 483 upregulated genes and 332 downregulated genes in corneas treated with R,R-RvD6, 58 upregulated genes and 36 downregulated genes were shared between R,R-RvD6 and RvD6 when compared to vehicle-treated corneas. This suggests that the signaling mechanisms underlying the biological activities of these two resolvins are distinct [34,37].

Further analysis revealed that R,R-RvD6 significantly decreased the induction of calcb, a gene encoding CGRP, and tac, which encodes SP—both peptides linked to pain [35], [48]–[51]. Additionally, the isomer, but not the RvD6 standard, enhanced the expression of trpm8, implying that R,R-RvD6 can modulate DELP (Fig. 3).

Fig. 3. Changes in expression of TG genes involved in pain post corneal injury.

TG were obtained 12 days after corneal injury and treatment with vehicle or RvD6si and analyzed by RNA-seq. Values are expressed as normalized counts. Modified from Pham TL et al. (Reference 35 in this review).

6. Conclusions

The biological impact of topically applied PEDF+DHA on injured corneas involves the generation of a novel RvD6 isomer, 4R-17R dihydroxy DHA, which displays robust biological repair properties and instigates a modulation of pain-responsive genes within the TG. Figure 4 provides a schematic overview of our current understanding regarding the effects of PEDF+DHA application on injured corneas. DHA is rapidly incorporated into membrane phospholipids within the corneal epithelium and is subsequently released following PEDF-induced activation of the PEDF-R with calcium-independent phospholipase A2 activity. The liberated intracellular DHA serves as a substrate for R,R-RvD6 synthesis, which is then released into tears. This newly identified lipid stimulates wound healing, tear secretion, corneal sensitivity, and nerve regeneration. The underlying mechanism involved changes in the TG transcriptome and the modulation of genes implicated in neuropathic pain. It’s important to note that treatment with PEDF or DHA alone fails to trigger these pathways.

Fig. 4. Scheme of R,R-RvD6 action in mouse cornea after refractive surgery.

Our investigations unequivocally establish that the bioactive lipid R,R-RvD6 holds considerable therapeutic potential for ocular conditions characterized by corneal nerve damage, dry eye, and associated pain.

Highlights.

• Corneal nerves are important to maintain tissue homeostasis.

• Discovery of a stereoisomer of resolvin D6 (R,R-RvD6) released in tears.

• R,R-RvD6 increases corneal sensitivity and nerve regeneration after damage.

• R,R-RvD6 stimulates genes in the trigeminal ganglia that modulate ocular pain.