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Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100033, January 01, 2021

Docosanoid signaling modulates corneal nerve regeneration: effect on tear secretion, wound healing, and neuropathic pain

Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1194/jlr.TR120000954

      Abstract

      The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using ω-3 supplementation show unclear and sometimes negative results. Using animal models of corneal nerve damage, we show that treating corneas with pigment epithelium-derived factor plus DHA increases nerve regeneration, wound healing, and tear secretion. The mechanism involves the activation of a calcium-independent phospholipase A2 that releases the incorporated DHA from phospholipids and enhances the synthesis of the docosanoids, neuroprotectin D1 (NPD1) and a new resolvin stereoisomer, resolvin D6i (RvD6i). NPD1 stimulates the synthesis of brain-derived neurotrophic factor, nerve growth factor, and semaphorin 7A. RvD6i treatment of injured corneas modulates gene expression in the TG resulting in enhanced neurogenesis, decreased neuropathic pain, and increased sensitivity. Taken together, these results represent a promising therapeutic option to reestablish the homeostasis of the cornea.

      Supplementary key words

      Abbreviations:

      BDNF (brain-derived neurotrophic factor), COX (cyclooxygenase), DE (dry eye), DED (dry eye disease), HDHA (hydroxy-DHA), LOX (lipoxygenase), NGF (nerve growth factor), NPD1 (neuroprotectin D1), PEDF (pigment epithelium-derived factor), PEDF-R (pigment epithelium-derived factor receptor), PRK (photorefractive keratectomy), RvD6 (resolvin D6), SP (substance P), TG (trigeminal ganglia)

      Cornea anatomy

      Figure thumbnail gr7
      Figure thumbnail gr1
      Fig. 1Corneal structure and innervation. A: The anatomy of human cornea after hematoxylin and eosin histological stain. All five layers are shown: epithelium, Bowman’s layer, stroma, Descemet’s layer, and endothelium. B: Whole mount view of complete human corneal epithelial nerve network obtained from the left eye of a 45-year-old male donor. C: Detailed course of epithelial nerve bundles running from the periphery to the convergence at the center of the cornea (B and C are reproduced with permission from Elsevier, Ref. 
      • He J.
      • Bazan N.G.
      • Bazan H.E.P.
      Mapping the entire human corneal nerve architecture.
      ).
      The epithelium consists of five to seven layers of nonkeratinized squamous epithelial cells, which are classified into three morphological cell types: superficial epithelial cells, intermediate wing cells, and the innermost basal epithelial cells with high rates of proliferation (
      • Meek K.M.
      • Knupp C.
      Corneal structure and transparency.
      ). The epithelial cells are connected by tight junctions that block the passage of foreign materials, such as dust, water, and bacteria, into the eye and provide a smooth surface that absorbs oxygen and cell nutrients. Moreover, the outermost layer of the epithelium is in contact with the tear film, which facilitates the moistness maintenance of the ocular surface that protects against damage from drying [dry eye (DE)]. Corneal epithelial cells regularly undergo a “turnover” with movement of stem cells from the limbal epithelium to the basal layer. These basal cells move toward the surface to generate two to three layers of wing cells and then begin terminal differentiation and desquamation. On average, the turnover time of human corneal epithelial cells is between 7 and 10 days (
      • Hanna C.
      • Bicknell D.S.
      • O’Brien J.E.
      Cell turnover in the adult human eye.
      ).
      The Bowman’s layer is a thin acellular layer that separates the epithelium from the stroma. It mainly contains collagen IV and laminin. The organization of these proteins is important to maintain the transparency of the tissue.
      The stroma layer is built up by quiescent keratocytes and a well-organized extracellular matrix composed primarily of highly ordered collagen type 1 fibrils, called lamella, and proteoglycans, and also constitutes the largest portion of the cornea (about 90% of corneal thickness). The stroma provides structural support to the cornea as well as transparency by facilitating the passage of light through collagen fibrils in a manner that prevents scattering. Keratocytes (the flat cells situated between collagen fibers) are the main cell residents of corneal stroma.
      The Descemet’s membrane is an acellular thin layer synthesized by the endothelium that is composed of fibronectin, laminin, and collagen IV and VII as well as proteoglycans. Damage to the Descemet’s membrane produces corneal edema and loss of vision.
      The last layer of the cornea is the endothelium, which is in contact with the aqueous humor. It is a monolayer of cells responsible for pumping fluid to regulate corneal stromal dehydration. Without endothelial pumps, there will be stroma edema, which produces opacity and decrease in vision. The human corneal endothelial cells have a very low capacity for proliferation, resulting in age-related reduction in cell density.
      An important characteristic of the cornea is its dense innervation (Fig. 1B). Most corneal nerve fibers are sensory in origin and derived mostly from neurons of the ophthalmic branch of the trigeminal ganglia (TG) (
      • Müller L.J.
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      • Kruse F.
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      Corneal nerves: structure, contents and function.
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      Mapping the entire human corneal nerve architecture.
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      • Said D.G.
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      Corneal nerves in health and disease.
      ). Anatomically, the corneal nerve network originates when stromal nerves enter the corneal sclera limbus in a radial fashion. To maintain corneal transparency, the arriving nerves lose their myelin sheaths and are surrounded by Schwann cells alone. In the stroma, the thick branches are divided into smaller nerve branches. Most of the branches penetrate the Bowman’s layer in the periphery and run to the center of the epithelium to form the epithelial nerve network (Fig. 1C), giving life to a dense network of nerve terminals.
      Corneal nerves stimulate tear secretion and blinking to maintain the integrity of the ocular surface (
      • Shaheen B.S.
      • Bakir M.
      • Jain S.
      Corneal nerves in health and disease.
      ). Alterations in corneal innervation occur in aging, diabetes, immunological diseases, such as rheumatoid arthritis and Sjögren’s syndrome, viral and bacterial infection, prolonged use of contact lenses, and refractive surgeries, such as laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) (
      • Cruzat A.
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      • Dana R.
      • Hamrah P.
      Inflammation and the nervous system: the connection in the cornea in patients with infectious keratitis.
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      Corneal nerve architecture in a donor with unilateral epithelial basement membrane dystrophy.
      ,
      • Pham T.L.
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      Mouse strains and sexual divergence in corneal innervation and nerve regeneration.
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      ,
      • He J.
      • Bazan H.E.P.
      Mapping the nerve architecture of diabetic human corneas.
      ,
      • Hamrah P.
      • Cruzat A.
      • Dastjerdi M.H.
      • Zheng L.
      • Shahatit B.M.
      • Bayhan H.A.
      • Dana R.
      • Pavan-Langston D.
      Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study.
      ). Complications from nerve damage diminish sensitivity, decrease tear secretion and blinking, and as a consequence, result in DE disease (DED) that produces neuropathic pain and corneal ulceration in severe cases. Due to the abundance of sensory nerves, the cornea is also a potent generator of pain in the human body.

      PEDF+DHA treatment for cornea-related damage: discovery of a resolvin D6 stereoisomer

      As mentioned, after damage, corneal nerve density slowly and incompletely recovered with decrease in sensitivity and DE symptoms. Early studies from our laboratory have shown that application of nerve growth factor (NGF) in conjunction with the ω-3 fatty acid DHA results in faster recovery of corneal nerve density after experimental PRK in rabbits (
      • Esquenazi S.
      • Bazan H.E.P.
      • Bui V.
      • He J.
      • Kim D.B.
      • Bazan N.G.
      Topical combination of NGF and DHA increases rabbit corneal nerve regeneration after photorefractive keratectomy.
      ). At that time, we proposed that the mechanisms could be mediated by the DHA-derived lipid mediator neuroprotectin D1 (NPD1), a docosanoid with potent anti-inflammatory and neuroprotective actions (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress.
      ). Synthesis of NPD1 in retinal pigment epithelial cells is stimulated by several growth factors with pigment epithelium-derived factor (PEDF) being 10 times more potent than NGF (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling.
      ). PEDF is a broad-acting neurotrophic and neuroprotective factor that regulates processes associated with angiogenesis, neuronal cell survival, and cell differentiation (
      • Tombran-Tink J.
      • Barnstable C.J.
      PEDF: a multifaceted neurotrophic factor.
      ) and is released from corneal epithelium after injury (
      • Kenchegowda S.
      • He J.
      • Bazan H.E.P.
      Involvement of pigment epithelium-derived factor, docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery.
      ). Posterior studies have shown that treatment with PEDF+DHA decreases inflammation and stimulates corneal wound healing and nerve regeneration in rabbit and mouse cornea models of experimental surgery, as well as in pathologies like diabetes and herpes simplex virus infection (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ,
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Recovery of corneal sensitivity, calcitonin gene-related peptide-positive nerves, and increased wound healing induced by pigment epithelial-derived factor plus docosahexaenoic acid after experimental surgery.
      ,
      • He J.
      • Cortina M.S.
      • Kakazu A.
      • Bazan H.E.P.
      The PEDF neuroprotective domain plus DHA induces corneal nerve regeneration after experimental surgery.
      ,
      • He J.
      • Neumann D.
      • Kakazu A.
      • Pham T.L.
      • Musarrat F.
      • Cortina M.S.
      • Bazan H.E.P.
      PEDF plus DHA modulate inflammation and stimulate nerve regeneration after HSV-1 infection.
      ,
      • He J.
      • Pham T.L.
      • Kakazu A.
      • Bazan H.E.P.
      Recovery of corneal sensitivity and increase in nerve density and wound healing in diabetic mice after PEDF plus DHA treatment.
      ). It is important to mention that the action requires treatment with both PEDF and DHA (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ). A 44-amino acid fragment of PEDF has neuroprotective activity, while an adjacent 34-amino acid peptide has anti-angiogenic activity (
      • Houenou L.J.
      • D’Costa A.P.
      • Li L.
      • Turgeon V.L.
      • Enyadike C.
      • Alberdi E.
      • Becerra S.P.
      Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons.
      ,
      • Amaral J.
      • Becerra S.P.
      Effects of human recombinant PEDF protein and PEDF-derived peptide 34-mer on choroidal neovascularization.
      ). Comparing the effect of the two peptides with the whole PEDF protein plus DHA in a rabbit model of corneal stroma dissection, we found that, unlike 34-mer-PEDF, 44 mer-PEDF+DHA decreases inflammation and increases tear secretion and corneal sensitivity and also promotes regeneration of corneal nerves by activating a PEDF receptor (PEDF-R) (
      • He J.
      • Cortina M.S.
      • Kakazu A.
      • Bazan H.E.P.
      The PEDF neuroprotective domain plus DHA induces corneal nerve regeneration after experimental surgery.
      ). This transmembrane receptor is expressed in the cornea and has calcium-independent phospholipase A2 (iPLAζ) activity (
      • Notari L.
      • Baladron V.
      • Aroca-Aguilar J.D.
      • Balko N.
      • Heredia R.
      • Meyer C.
      • Notario P.M.
      • Saravanamuthu S.
      • Nueda M.-L.
      • Sanchez-Sanchez F.
      • et al.
      Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor.
      ,
      • Pham T.L.
      • He J.
      • Kakazu A.H.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.
      ) that releases DHA, which is enriched in the sn-2 position of membrane phospholipids by DHA supplementation.
      Early studies on calf corneas identified PC, PE, and sphingomyelin as the main phospholipids in the tissue (
      • Broekhuyse R.M.
      Phospholipids in tissues of the eye. I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids.
      ). Among these phospholipids, PC is the most abundant with the highest content in the epithelium. Similar observations were reported in human (
      • Tschetter R.T.
      Lipid analysis of the human cornea with and without arcus senilis.
      ) and rabbit corneas (
      • Bazan H.E.
      • Bazan N.G.
      Composition of phospholipids and free fatty acids and incorporation of labeled arachidonic acid in rabbit cornea. Comparison of epithelium, stroma and endothelium.
      ). In the rabbit, oleic acid (18:1) is the dominant fatty acid esterified in phospholipids in all of the corneal layers (about 50% of total fatty acids in phospholipids) followed by palmitic acid (16:0), which comprises about 16–18%. With respect to the PUFAs esterified in phospholipids, the higher percentage (about 9% of total fatty acids) corresponds to AA, while the percentage of EPA and DHA esterified in phospholipids is much lower (around 1.6% of total fatty acids) (
      • Bazan H.E.
      • Bazan N.G.
      Composition of phospholipids and free fatty acids and incorporation of labeled arachidonic acid in rabbit cornea. Comparison of epithelium, stroma and endothelium.
      ).
      DHA topical treatment of mouse corneas, in which stromal nerves had been damaged, produced a rapid incorporation of the fatty acid in PC and PE molecular species containing 18:1-DHA (
      • Pham T.L.
      • He J.
      • Kakazu A.H.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.
      ), demonstrating that the addition of the PUFAs created a significant enrichment of DHA in the lipid membrane composition (Fig. 2A).
      Figure thumbnail gr2
      Fig. 2Incorporation of DHA into PC and PE after 1 h of DHA topical treatment to corneas of mice with damaged stromal nerves. A: Mouse corneas were injured and topically treated with DHA for 1 h and then lipids extracted and analyzed by LC-MS/MS (
      • Pham T.L.
      • He J.
      • Kakazu A.H.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.
      ). The proportion of PC and PE containing oleic acid (18:1) in the sn-1 position and DHA in the sn-2 position. PE was more enriched in DHA than PC. B: Release of DHA and synthesis of the monohydroxy-DHA derivatives after corneal injury and topical treatment with PEDF+DHA for 3 h. Corneal lipid profiles were analyzed by MS-based lipidomic analysis. ∗P < 0.05 with the t test statistical analysis to compare two groups at 95% of the confidence level.
      Tissue damage activates phospholipase A2 that releases PUFAs, such as AA, EPA, and DHA, from the sn-2 position (
      • Katsura K.
      • Rodriguez de Turco E.B.
      • Siesjö B.K.
      • Bazan N.G.
      Effects of hyperglycemia and hypercapnia on lipid metabolism during complete brain ischemia.
      ,
      • Rodriguez de Turco E.B.
      • Belayev L.
      • Liu Y.
      • Busto R.
      • Parkins N.
      • Bazan N.G.
      • Ginsberg M.D.
      Systemic fatty acid responses to transient focal cerebral ischemia: influence of neuroprotectant therapy with human albumin.
      ). Several early studies from our laboratory and others have demonstrated that the cornea responds to injury, increasing the synthesis of prostaglandins by activation of cyclooxygenase (COX)-2 (
      • Bazan H.E.
      • Birkle D.L.
      • Beuerman R.
      • Bazan N.G.
      Cryogenic lesion alters the metabolism of arachidonic acid in rabbit cornea layers.
      ,
      • Bazan H.E.
      • Tao Y.
      • DeCoster M.A.
      • Bazan N.G.
      Platelet-activating factor induces cyclooxygenase-2 gene expression in corneal epithelium. Requirement of calcium in the signal transduction pathway.
      ,
      • Liclican E.L.
      • Nguyen V.
      • Sullivan A.B.
      • Gronert K.
      Selective activation of the prostaglandin E2 circuit in chronic injury-induced pathologic angiogenesis.
      ,
      • Amico C.
      • Yakimov M.
      • Catania M.V.
      • Giuffrida R.
      • Pistone M.
      • Enea V.
      Differential expression of cyclooxygenase-1 and cyclooxygenase-2 in the cornea during wound healing.
      ) and HETEs and lipoxin A4 (LxA4) by activation of lipoxygenases (LOXs) (
      • Hurst J.S.
      • Balazy M.
      • Bazan H.E.
      • Bazan N.G.
      The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury.
      ,
      • Sharma G.D.
      • Ottino P.
      • Bazan N.G.
      • Bazan H.E.P.
      Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase Calpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis.
      ,
      • Leedom A.J.
      • Sullivan A.B.
      • Dong B.
      • Lau D.
      • Gronert K.
      Endogenous LXA4 circuits are determinants of pathological angiogenesis in response to chronic injury.
      ). Because the concentration of DHA in membrane lipids is very low (Fig. 2A) (
      • Bazan H.E.
      • Bazan N.G.
      Composition of phospholipids and free fatty acids and incorporation of labeled arachidonic acid in rabbit cornea. Comparison of epithelium, stroma and endothelium.
      ), we found that the addition of DHA to the corneas treated with PEDF was important to increasing the synthesis of lipid derivatives of DHA (docosanoids) with strong anti-inflammatory properties (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ,
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ,
      • Cortina M.S.
      • He J.
      • Russ T.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 restores corneal nerve integrity and function after damage from experimental surgery.
      ). Therefore, activating the calcium-independent phospholipase A2 (iPLA2ζ) of the PEDF-R by treating the corneas with PEDF+DHA leads to a more than 3,000-fold increase of free DHA released from the cornea (Fig. 2B).
      Free DHA is then the substrate for the synthesis of 14- and 17-hydroperoxy-DHA that are rapidly converted in the more stable hydroxy-DHA (HDHA) derivatives (Fig. 2B). These results confirmed our hypothesis that PEDF+DHA treatment stimulates the formation of docosanoids derived from DHA.
      Figure 3 shows a scheme of bioactive lipids resulting from AA, EPA, and DHA. While many AA lipid mediators, as well as some EPA lipid mediators, have strong pro-inflammatory properties, all known DHA mediators (the docosanoids) act to protect and resolve inflammation (
      • Bazan N.G.
      Docosanoids and elovanoids from omega-3 fatty acids are pro-homeostatic modulators of inflammatory responses, cell damage and neuroprotection.
      ,
      • Serhan C.N.
      • Levy B.D.
      Resolvins in inflammation: emergence of the pro- resolving superfamily of mediators.
      ). They constitute part of a family named specialized pro-resolvin mediators that includes NPD1 and other protectins, maresins, and resolvins of the D series (
      • Serhan C.N.
      • Levy B.D.
      Resolvins in inflammation: emergence of the pro- resolving superfamily of mediators.
      ) and the newer sulfide conjugates of protectins (PCTRs), maresins (MCTRs), and resolvins (RCTRs). The synthetic mechanism to produce the specialized pro-resolvin mediators involves LOXs (including 15-LOX as primary catalyzer and 5-LOX as secondary catalyzer), COX (in the presence of aspirin), and cytochrome P450 enzymes (
      • Krishnamoorthy S.
      • Recchiuti A.
      • Chiang N.
      • Yacoubian S.
      • Lee C.-H.
      • Yang R.
      • Petasis N.A.
      • Serhan C.N.
      Resolvin D1 binds human phagocytes with evidence for proresolving receptors.
      ). Information about the signaling mechanisms of DHA lipid mediators is still limited, especially identification of their receptors (Table 1). Most of the known receptors belong to the family of G protein-coupled receptors. In addition, some docosanoids share the same receptor, but their activation exerts specific biological activities (
      • Serhan C.N.
      • Levy B.D.
      Resolvins in inflammation: emergence of the pro- resolving superfamily of mediators.
      ).
      Figure thumbnail gr3
      Fig. 3Lipid mediators derived from the three most abundant essential fatty acids, AA, EPA, and DHA, esterified in the sn-2 position of the phospholipids. Depending on the primary catalyzing enzyme, COX-2, and 5- and 15-LOXs, there is synthesis of a variety of bioactive lipids involved in inflammation as well as in resolution of the inflammatory response. Mediators from AA are highlighted in orange, EPA in green, and DHA in blue.
      Table 1List of reported receptors of docosanoids
      NameReceptorsReferencesExpression in the Cornea
      Resolvin D1ALX/FPR2, GPR32 (DRV1)(
      • Krishnamoorthy S.
      • Recchiuti A.
      • Chiang N.
      • Yacoubian S.
      • Lee C.-H.
      • Yang R.
      • Petasis N.A.
      • Serhan C.N.
      Resolvin D1 binds human phagocytes with evidence for proresolving receptors.
      )
      Yes
      Resolvin D2GPR18 (DRV2)(
      • Chiang N.
      • Dalli J.
      • Colas R.A.
      • Serhan C.N.
      Identification of resolvin D2 receptor mediating resolution of infections and organ protection.
      )
      ND
      Resolvin D3ALX/FPR2, GPR32 (DRV1)(
      • Dalli J.
      • Winkler J.W.
      • Colas R.A.
      • Arnardottir H.
      • Cheng C.-Y.C.
      • Chiang N.
      • Petasis N.A.
      • Serhan C.N.
      Resolvin D3 and aspirin-triggered resolvin D3 are potent immunoresolvents.
      )
      Yes
      Resolvin D4N/A
      Resolvin D5GPR32(
      • Chiang N.
      • Fredman G.
      • Bäckhed F.
      • Oh S.F.
      • Vickery T.
      • Schmidt B.A.
      • Serhan C.N.
      Infection regulates pro-resolving mediators that lower antibiotic requirements.
      )
      ND
      Resolvin D6N/A
      Neuroprotectin D1GPR37 (Pael-R)(
      • Bang S.
      • Xie Y.-K.
      • Zhang Z.-J.
      • Wang Z.
      • Xu Z.-Z.
      • Ji R.-R.
      GPR37 regulates macrophage phagocytosis and resolution of inflammatory pain.
      )
      ND
      Maresin 1LGR6(
      • Chiang N.
      • Libreros S.
      • Norris P.C.
      • de la Rosa X.
      • Serhan C.N.
      Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions.
      )
      ND
      Maresin 2N/A
      ND, not determined; N/A, not available.
      Recently, we discovered a novel docosanoid, a stereoisomer of resolvin D6 (RvD6), named RvD6i (Fig. 4) , that is released in mouse tears after injury and treatment with PEDF+DHA (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). The fragmentation pattern of this new lipid shows at least six matched product ions that coincide with RvD6. Resolvin D6 had been found in some tissues, and studies in plasma from healthy individuals showed that RvD6 is a biomarker that decreases with aging (
      • Jové M.
      • Maté I.
      • Naudí A.
      • Mota-Martorell N.
      • Portero-Otín M.
      • De la Fuente M.
      • Pamplona R.
      Human aging is a metabolome-related matter of gender.
      ). RvD6 is also released from stem cells isolated from human periodontal ligaments, which is important in tissue regeneration (
      • Cianci E.
      • Recchiuti A.
      • Trubiani O.
      • Diomede F.
      • Marchisio M.
      • Miscia S.
      • Colas R.A.
      • Dalli J.
      • Serhan C.N.
      • Romano M.
      Human periodontal stem cells release specialized proresolving mediators and carry immunomodulatory and prohealing properties regulated by lipoxins.
      ). However, RvD6 is not detected in normal human tears (
      • English J.T.
      • Norris P.C.
      • Hodges R.R.
      • Dartt D.A.
      • Serhan C.N.
      Identification and profiling of specialized pro-resolving mediators in human tears by lipid mediator metabolomics.
      ). Compared with treatments with PEDF+DHA and RvD6, the new RvD6i accelerated corneal wound healing and sensitivity, demonstrating a higher bioactivity (Fig. 4A, B).
      Figure thumbnail gr4
      Fig. 4Structure of the RvD6i. The new isomer was synthesized after topical stimulation of mouse injured corneas with PEDF+DHA and released in tears. It was analyzed by LC-MS/MS and showed at least six matched daughter ions with an RvD6 standard but with an earlier retention time (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). Posterior studies show that the peak retention time coincides with chemically synthetized R,R-RvD6i in a chiral column (unpublished observations).

      Use of DHA for DED

      DED affects between 5% and 40% of adults older than 40 years (
      2007. The epidemiology of dry eye disease: report of the Epidemiology Subcommittee of the International Dry Eye WorkShop.
      ,
      • Stapleton F.
      • Alves M.
      • Bunya V.Y.
      • Jalbert I.
      • Lekhanont K.
      • Malet F.
      • Na K.-S.
      • Schaumberg D.
      • Uchino M.
      • Vehof J.
      • et al.
      TFOS DEWS II epidemiology report.
      ) with an estimated 16.4 million people impacted in the United States (
      • Farrand K.F.
      • Fridman M.
      • Stillman I.Ö.
      • Schaumberg D.A.
      Prevalence of diagnosed dry eye disease in the united states among adults aged 18 years and older.
      ). In a recent Dry Eye Workshop (DEWS II), DE was defined as “a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tearfilm, and accompanied by ocular symptoms, in which tearfilm instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles.” (Ref. 
      • Stapleton F.
      • Alves M.
      • Bunya V.Y.
      • Jalbert I.
      • Lekhanont K.
      • Malet F.
      • Na K.-S.
      • Schaumberg D.
      • Uchino M.
      • Vehof J.
      • et al.
      TFOS DEWS II epidemiology report.
      ; p. 278).
      Within the last decade, there have been a number of clinical trials of DED patients with different etiologies using ω-3 fatty acid, DHA and EPA, supplementation with the argument that dietary fatty acids can be incorporated in the lacrimal gland as well as in plasma phospholipids (
      • Schnebelen C.
      • Viau S.
      • Grégoire S.
      • Joffre C.
      • Creuzot-Garcher C.P.
      • Bron A.M.
      • Bretillon L.
      • Acar N.
      Nutrition for the eye: different susceptibility of the retina and the lacrimal gland to dietary omega-6 and omega-3 polyunsaturated fatty acid incorporation.
      ). However, the effect of oral PUFA supplementation in DED is controversial. While some studies showed improvement, others showed insignificant effects. In Table 2, we summarized clinical trials conducted in the last 10 years in which supplementation with DHA was used to treat DED of different etiologies.
      Table 2Summary of clinical trials in the last 10 years for DED using ω-3 fatty acid treatment
      StudyNumber of Patients/TreatmentRandomized/ControlledMaskingEffectComments
      Brignole- Baudouin et al., 2011 (
      • Brignole-Baudouin F.
      • Baudouin C.
      • Aragona P.
      • Rolando M.
      • Labetoulle M.
      • Pisella P.J.
      • Barabino S.
      • Siou-Mermet R.
      • Creuzot-Garcher C.
      A multicentre, double- masked, randomized, controlled trial assessing the effect of oral supplementation of omega-3 and omega-6 fatty acids on a conjunctival inflammatory marker in dry eye patients.
      )
      DE, n = 127, time = 90 days

      Group 1, n = 61

      142.5 mg EPA, 95 mg DHA, and supplements, three times daily

      Group 2, n = 66

      Placebo, medium-chain triglyceride, three times daily
      Yes/YesDoubleNo significant effectOnly decrease in the percentage of HLA-DR-positive cell was detected in treated group
      Wojtowicz et al., 2011 (
      • Wojtowicz J.C.
      • Butovich I.
      • Uchiyama E.
      • Aronowicz J.
      • Agee S.
      • McCulley J.P.
      Pilot, prospective, randomized, double-masked, placebo-controlled clinical trial of an omega-3 supplement for dry eye.
      )
      DE, n = 36, time = 90 days

      Group 1

      450 mg EPA, 300 mg DHA, and 1,000 mg flaxseed oil, one time daily

      Group 2

      Placebo, wheat germ oil
      Yes/YesDoubleNo significant effectNo changes in aqueous tear evaporation
      Bhargava et al., 2013 (
      • Bhargava R.
      • Kumar P.
      • Kumar M.
      • Mehra N.
      • Mishra A.
      A randomized controlled trial of omega-3 fatty acids in dry eye syndrome.
      )
      DE, n = 528, time = 3 months

      Group 1, n = 264

      325 mg EPA and 175 mg DHA, two times daily

      Group 2, n = 254

      Placebo, two times daily
      Yes/YesDoubleImproved
      Kangari et al., 2013 (
      • Kangari H.
      • Eftekhari M.H.
      • Sardari S.
      • Hashemi H.
      • Salamzadeh J.
      • Ghassemi-Broumand M.
      • Khabazkhoob M.
      Short-term consumption of oral omega-3 and dry eye syndrome.
      )
      DE, n = 64, time = 30 days

      Group 1, n = 33

      180 mg EPA and 120 mg DHA, two times daily

      Group 2, n = 31

      Placebo, medium-chain triglyceride
      YesDoubleSlightly improved
      Oleñik et al., 2013 (
      • Oleñik A.
      • Jiménez-Alfaro I.
      • Alejandre-Alba N.
      • Mahillo-Fernández I.
      A randomized, double-masked study to evaluate the effect of omega-3 fatty acids supplementation in meibomian gland dysfunction.
      )
      Meibomian gland dysfunction, n = 64, time = 3 months

      Group 1, n = 33

      Brudysec (350 mg DHA, 42,5 mg EPA, 30 mg DPA), three times daily

      Group 2, n = 31

      Placebo, 500 mg sunflower oil, three times daily. All patients received cleaning the lid margins with neutral baby shampoo and artificial tears without preservatives
      Yes/NoDoubleSlightly improvedNo significant differences in corneal staining from placebo
      Ong et al., 2013 (
      • Ong N.H.
      • Purcell T.L.
      • Roch-Levecq A.-C.
      • Wang D.
      • Isidro M.A.
      • Bottos K.M.
      • Heichel C.W.
      • Schanzlin D.J.
      Epithelial healing and visual outcomes of patients using omega-3 oral nutritional supplements before and after photorefractive keratectomy: a pilot study.
      )
      Healthy PRK patients, n = 18, time = 6 weeks

      Group 1, n = 9

      250 mg EPA and DHA, 333 mg flaxseed oil, and 61 IU vitamin E, three times daily

      Group 2, n = 9

      Control
      Yes/YesSingleImprovedTreatment was 2 weeks before PRK surgery through 1 month after surgery
      Sheppard et al., 2013 (
      • Sheppard J.D.
      • Singh R.
      • McClellan A.J.
      • Weikert M.P.
      • Scoper S.V.
      • Joly T.J.
      • Whitley W.O.
      • Kakkar E.
      • Pflugfelder S.C.
      Long-term supplementation with n-6 and n-3 pufas improves moderate-to-severe keratoconjunctivitis sicca: a randomized double-blind clinical trial.
      )
      Post-menopausal women with DE, n = 38, time = 6 months

      Group 1, n = 19

      49 mg ALA, 31.5 mg EPA, 3.75 mg DPA, 25 mg DHA, 177.5 mg LA, 60 mg GLA, <0.75 AA, and supplements, four times daily

      Group 2, n = 19

      Placebo
      Yes/YesDoubleImprovedPlacebo treatment also increased HLA-DR intensity by 36 ± 9% and CD11c by 34 ± 7% when compared with supplement treatment
      Oleñik, 2014 (
      • Oleñik A.
      Effectiveness and tolerability of dietary supplementation with a combination of omega-3 polyunsaturated fatty acids and antioxidants in the treatment of dry eye symptoms: Results of a prospective study.
      )
      DE, n = 905, time = 12 weeks

      Brudysec (350 mg DHA, 42,5 mg EPA, 30 mg DPA), three times daily.

      No control of placebo
      No/NoNoImprovedA total of 68.1% of patients reported better tolerance to contact lenses after treatment
      Georgakopoulos et al., 2017 (
      • Georgakopoulos C.D.
      • Makri O.E.
      • Pagoulatos D.
      • Vasilakis P.
      • Peristeropoulou P.
      • Kouli V.
      • Eliopoulou M.I.
      • Psachoulia C.
      Effect of omega-3 fatty acids dietary supplementation on ocular surface and tear film in diabetic patients with dry eye.
      )
      Diabetic patients with DE, time = 3 months

      Group 1, n = 36

      170 mg EPA and 115 mg DHA, three times daily
      No/NoNoImproved
      Bhargava et al., 2015 (
      • Bhargava R.
      • Kumar P.
      • Phogat H.
      • Kaur A.
      • Kumar M.
      Oral omega-3 fatty acids treatment in computer vision syndrome related dry eye.
      )
      Computer-related DE, n = 456, time = 3 months

      Group 1, n = 220

      180 mg EPA and 120 mg DHA, two times daily

      Group 2, n = 236

      Placebo containing olive oil, two times daily

      Baseline (T0), 1 month of treatment (T1), 2 months of treatment (T2), 3 months of treatment (T3)
      Yes/YesDoubleImproved
      Deinema et al., 2017 (
      • Deinema L.A.
      • Vingrys A.J.
      • Wong C.Y.
      • Jackson D.C.
      • Chinnery H.R.
      • Downie L.E.
      A randomized, double-masked, placebo-controlled clinical trial of two forms of omega-3 supplements for treating dry eye disease.
      )
      DE, n = 54, time = 3 months

      Group 1, n = 18

      Krill oil (945 mg/day EPA + 510 mg/day DHA)

      Group 2, n = 19

      Fish oil (1,000 mg/day EPA + 500 mg/day DHA)

      Group 3, n = 17

      Placebo (olive oil, 1,500 mg/day)
      Yes/YesDoubleSlightly improvedBoth krill and fish oil moderately reduced the DE symptoms. The pro-inflammatory cytokine, interleukin 17A, was significantly reduced in the krill oil group only at day 90
      Goyal et al., 2017 (
      • Goyal P.
      • Jain A.K.
      • Malhotra C.
      Oral omega-3 fatty acid supplementation for laser in situ keratomileusis-associated dry eye.
      )
      LASIK patients, n = 60, time = 12 weeks

      Group 1, n = 30

      180 mg EPA and 120 mg DHA, four times daily

      Group 2, n = 3

      Placebo
      Yes/YesDoubleSlightly improvedFewer eyes had conjunctival staining with Lissamine
      DREAM, 2019 (
      • Asbell P.A.
      • Maguire M.G.
      • Pistilli M.
      • Ying G-s.
      • Szczotka-Flynn L.B.
      • Hardten D.R.
      • Lin M.C.
      • Shtein R.M.
      Dry Eye Assessment and Management Study Research Group
      n-3 Fatty acid supplementation for the treatment of dry eye disease.
      )
      DE, n = 499, time = 12 months

      Group 1, n = 329

      400 mg EPA and 200 mg DHA, five times daily

      Group 2, n = 170

      Placebo, 1,000 mg refined olive oil, five times daily
      Yes/YesDoubleNo significant effectSignificantly increased EPA and DHA in red blood cells
      Fogt et al., 2019 (
      • Fogt J.S.
      • Fogt N.
      • King-Smith P.E.
      • Liu H.
      • Barr J.T.
      Changes in tear lipid layer thickness and symptoms following the use of artificial tears with and without omega-3 fatty acids: a randomized, double-masked, crossover study.
      )
      DE, n = 19, time = 1 h

      Drug 1, n = 19

      Refresh Optive plus Omega-3, flaxseed oil

      Drug 2, n = 19

      Refresh Optive

      The drug is randomly picked for two different visits (>2 days between)
      Yes/YesDoubleImproved (short time)The lipid layer thickness (LLT) was increased from baseline at 15 min for both treatments. Only Refresh Optive plus Omega-3 patients had higher LLT up to 1 h after instillation
      Bold type indicates the clinical trial using topical eye drops. LASIK, laser in situ keratomileusis.
      One of the most important trials, the DREAM study, which involved a total of 499 patients with 329 receiving 12 months of supplementation with EPA and DHA and 170 patients treated with refined olive oil as a placebo (
      • Asbell P.A.
      • Maguire M.G.
      • Pistilli M.
      • Ying G-s.
      • Szczotka-Flynn L.B.
      • Hardten D.R.
      • Lin M.C.
      • Shtein R.M.
      Dry Eye Assessment and Management Study Research Group
      n-3 Fatty acid supplementation for the treatment of dry eye disease.
      ), suggested that there was no improvement. This study increases the doubtfulness about the benefit of DHA in the treatment of DED. For this reason, in this review, we point out problems that may explain the controversial results of DHA supplementation.
      One concern is the form of DHA supplementation. Most of the studies employed natural enriched fish oil. However, analysis of fish oil composition showed that the PUFAs are mainly esterified in triglycerides. DHA from the diet needs to be taken up by the liver before being esterified in the sn-2 position of membrane phospholipid, mainly PC (
      • Polozova A.
      • Salem N.
      Role of liver and plasma lipoproteins in selective transport of n-3 fatty acids to tissues: a comparative study of 14C-DHA and 3H-oleic acid tracers.
      ). DHA-phospholipids are then packaged in VLDLs or other lipoproteins before being released into the blood stream (
      • Polozova A.
      • Salem N.
      Role of liver and plasma lipoproteins in selective transport of n-3 fatty acids to tissues: a comparative study of 14C-DHA and 3H-oleic acid tracers.
      ,
      • Bazan N.G.
      • Molina M.F.
      • Gordon W.C.
      Docosahexaenoic acid signalolipidomics in nutrition: significance in aging, neuroinflammation, macular degeneration, Alzheimer’s, and other neurodegenerative diseases.
      ). Therefore, the possibility that supplementation of DHA or EPA from fish oil reaches the ocular surface, especially the cornea, is very low. This is supported by previous studies where krill oil, which mainly contains PC with long chain PUFAs, showed a higher absorption rate in rat blood and brain than fish oil (
      • Ahn S.H.
      • Lim S.J.
      • Ryu Y.M.
      • Park H.-R.
      • Suh H.J.
      • Han S.H.
      Absorption rate of krill oil and fish oil in blood and brain of rats.
      ). There is only one study that uses krill oil to treat DED, a small clinical trial (18 participants per group) in which Deinema and colleagues showed lower Ocular Surface Disease Index and IL-17A levels in krill oil supplementation than in fish oil after 90 days of treatment (
      • Deinema L.A.
      • Vingrys A.J.
      • Wong C.Y.
      • Jackson D.C.
      • Chinnery H.R.
      • Downie L.E.
      A randomized, double-masked, placebo-controlled clinical trial of two forms of omega-3 supplements for treating dry eye disease.
      ) and Table 2.
      In addition, it is important to note that the cornea is avascular, therefore, the probability that dietary fatty acids are incorporated into the corneal cellular membrane is limited. This is supported by a study using 14C-labeled DHA given orally to rats, which showed a very small amount (less than 0.03% of the oral dose) of DHA that reached the eye compartment (
      • Graf B.A.
      • Duchateau G.S.M.J.E.
      • Patterson A.B.
      • Mitchell E.S.
      • van Bruggen P.
      • Koek J.H.
      • Melville S.
      • Verkade H.J.
      Age dependent incorporation of 14C-DHA into rat brain and body tissues after dosing various 14C-DHA-esters.
      ). Of this quantity, the amount that might get into the cornea is very low because the retina takes most of the DHA from subretinal blood vessels. Therefore, PUFA enrichment in the lacrimal gland is insufficient to ensure a beneficial treatment in the cornea.
      To our knowledge, there is only one clinical trial using topical DHA (
      • Fogt J.S.
      • Fogt N.
      • King-Smith P.E.
      • Liu H.
      • Barr J.T.
      Changes in tear lipid layer thickness and symptoms following the use of artificial tears with and without omega-3 fatty acids: a randomized, double-masked, crossover study.
      ) (Table 2). This trial was based on previous studies showing that AA, DHA, and EPA were found in the tears of patients with DED and that the ratio of ω-6 (AA):ω-3 (DHA+EPA) correlates with the severity of the tear film dysfunction (
      • Walter S.D.
      • Gronert K.
      • McClellan A.L.
      • Levitt R.C.
      • Sarantopoulos K.D.
      • Galor A.
      ω-3 Tear film lipids correlate with clinical measures of dry eye.
      ). The small trial (19 patients treated topically with DHA) demonstrated that treatment with eye drops containing omega-3 fatty acids increases lipid layer thickness of the tear film up to 1 h after instillation (
      • Fogt J.S.
      • Fogt N.
      • King-Smith P.E.
      • Liu H.
      • Barr J.T.
      Changes in tear lipid layer thickness and symptoms following the use of artificial tears with and without omega-3 fatty acids: a randomized, double-masked, crossover study.
      ).
      Lastly, our animal studies show that DHA is rapidly incorporated in the corneal phospholipids, mainly in PE and PC, to increase nerve density. A decrease in nerve density is a well-documented alteration in DED that requires both PEDF and DHA to regenerate the nerves. The treatment releases DHA and stimulates the synthesis of RvD6i, and this docosanoid increases wound healing and sensitivity (Fig. 5A, B) and could be of better therapeutic use than DHA for DED (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ).
      Figure thumbnail gr5
      Fig. 5RvD6i accelerates corneal wound healing and sensitivity. A: Representative images of mouse cornea wounded area stained with methylene blue after 20 h of an injury that damaged the epithelial and anterior stroma nerves. The animals received eye drops containing PEDF+DHA or RvD6i in similar concentrations three times per day. The images were taken with a dissecting microscope and quantified using Photoshop software (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). B: Recovery of cornea sensitivity at 3, 6, and 9 days after injury and treatment with PEDF+DHA or RvD6i (three times per day) using a noncontact aesthesiometer. RvD6i-treated mice recovered sensitivity sooner than PEDF+DHA-treated corneas. C: Expression of genes involved in inflammation and pain in the TG of RvD6i topically treated corneas. TG were obtained 12 days after cornea injury and treatment with RvD6i and analyzed by RNA sequencing (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). Calcb and Tac1 genes were downregulated while Trpm8 and Rictor genes were upregulated in the TG neurons by cornea treatment with RvD6i. ∗P < 0.05 with the t test statistical analysis to compare two groups at 95% of the confidence level.
      The effectiveness of docosanoids in decreasing inflammation and increasing corneal wound healing, nerve regeneration, and tear secretion has been demonstrated clearly on several different models of injury, infection, diabetes, corneal angiogenesis, and transplantation (Table 3). These results emphasized the action of docosanoids as potent drugs.
      Table 3In vivo studies using PEDF+DHA or docosanoids for corneal damage
      AnimalModelDocosanoidsAdministrationKey Result
      MouseCorneal epithelium removal up to the corneal/limbal border (
      • Gronert K.
      • Maheshwari N.
      • Khan N.
      • Hassan I.R.
      • Dunn M.
      • Schwartzman M.L.
      A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense.
      )
      NPD1, 17S-HDHATopical eye drops, three times daily for 96 hIncreased the rate of re-epithelialization. Increased PMNs in the cornea. Decreased formation of the pro-inflammatory chemokine KC
      MouseSuture-induced inflammatory corneal angiogenesis (
      • Jin Y.
      • Arita M.
      • Zhang Q.
      • Saban D.R.
      • Chauhan S.K.
      • Chiang N.
      • Serhan C.N.
      • Dana R.
      Anti-angiogenesis effect of the novel anti-inflammatory and pro-resolving lipid mediators.
      )
      RvD1Subconjunctival injection every 48 h; time = 14 daysReduced numbers of infiltrating neutrophils and macrophages and reduced mRNA expression levels of TNF-α, IL-1α, IL-1β, VEGF-A, VEGF-C, and VEGFR2. Suppressed suture-induced or IL-1β-induced hemangiogenesis but not lymphangiogenesis
      RabbitExperimental PRK (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ,
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Recovery of corneal sensitivity, calcitonin gene-related peptide-positive nerves, and increased wound healing induced by pigment epithelial-derived factor plus docosahexaenoic acid after experimental surgery.
      ,
      • He J.
      • Cortina M.S.
      • Kakazu A.
      • Bazan H.E.P.
      The PEDF neuroprotective domain plus DHA induces corneal nerve regeneration after experimental surgery.
      )
      PEDF+DHA, PEDF domains + DHATopical using collagen shield, twice a week; time = 8 weeksIncreased nerve density and tear secretion in treated group for 8 weeks. with PEDF+DHA. NPD1 synthesis peaked at 1 week and was four times higher in the PEDF+DHA-treated group than in the controls. The 44-mer domain of PEDF is more potent than the 34-mer domain
      RabbitExperimental PRK (
      • Cortina M.S.
      • He J.
      • Russ T.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 restores corneal nerve integrity and function after damage from experimental surgery.
      )
      NPD1Topical eye drops of NPD1 (33 ng/eye) three times daily for 6 weeksIncreased subepithelial corneal nerves and tear secretion. Decreased neutrophil infiltration after 2 and 4 days of treatment
      MouseCorneal allotransplantation (
      • Hua J.
      • Jin Y.
      • Chen Y.
      • Inomata T.
      • Lee H.
      • Chauhan S.K.
      • Petasis N.A.
      • Serhan C.N.
      • Dana R.
      The resolvin D1 analogue controls maturation of dendritic cells and suppresses alloimmunity in corneal transplantation.
      )
      RvD1 analogIntravenous injectionReduced allosensitization. Reduced angiogenesis at the graft site. Enhanced graft survival
      MouseType 2 diabetes (
      • Obrosov A.
      • Coppey L.J.
      • Shevalye H.
      • Yorek M.A.
      Effect of fish oil vs. resolvin D1, E1, methyl esters of resolvins D1 or D2 on diabetic peripheral neuropathy.
      )
      RvD1, RvD1-methyl ester, RvD2-methyl esterDaily intraperitoneal injections of 1 ng/g body weight for 8 weeksReduced the diabetes-induced corneal nerve lost. Methyl ester version is less bioactive than free fatty acid
      RabbitHSV1 corneal infection (
      • He J.
      • Neumann D.
      • Kakazu A.
      • Pham T.L.
      • Musarrat F.
      • Cortina M.S.
      • Bazan H.E.P.
      PEDF plus DHA modulate inflammation and stimulate nerve regeneration after HSV-1 infection.
      )
      PEDF+DHATopical eye drops, three times daily for 2 weeks. Topical using collagen shield, twice a week for 10 weeks moreStronger infiltration of CD4+T cells, neutrophils, and macrophages at 7 days, then decreased by 14 days. Corneal nerve density increased at 12 weeks with functional recovery of corneal sensation
      MouseType 1 diabetes. Corneal epithelium removal inside 2 mm diameter central area (
      • He J.
      • Pham T.L.
      • Kakazu A.
      • Bazan H.E.P.
      Recovery of corneal sensitivity and increase in nerve density and wound healing in diabetic mice after PEDF plus DHA treatment.
      )
      PEDF+DHATopical eye drops, three times daily for 14 daysIncrease in corneal epithelial nerve regeneration, SP-positive nerve density and tear volume. Accelerated corneal wound healing, selectively recruited type 2 macrophages, and prevented neutrophil infiltration
      MouseCorneal nerve cutting (
      • Pham T.L.
      • He J.
      • Kakazu A.H.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.
      )
      PEDF+DHATopical eye drops, three times daily for 7 daysIncreased nerve regeneration and tear secretion. Phospholipase A2 activity of the PEDF-R is required for the working mechanism
      MouseType 1 diabetes. Corneal epithelium removal inside 2 mm diameter central area (
      • Zhang Z.
      • Hu X.
      • Qi X.
      • Di G.
      • Zhang Y.
      • Wang Q.
      • Zhou Q.
      Resolvin D1 promotes corneal epithelial wound healing and restoration of mechanical sensation in diabetic mice.
      )
      RvD1Topical eye drops, four times daily for 14 daysPromotes corneal epithelial wound healing and nerve regeneration
      MouseCorneal epithelium removal inside 2 mm diameter central area (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      )
      RvD6iTopical eye drops, three times daily for 12 daysDiscovered the RvD6i underlying the mechanism of PEDF+DHA. Increased corneal wound healing, sensitivity, and nerve regeneration. Reduced inflammatory- and pain-related neuropeptides, increased ion channel gene expression in TG
      Bold type indicates studies from our laboratory. HSV1, herpes simplex virus.

      RvD6i regulates genes involved in neurogenesis and pain in the TG

      Previous studies have showed that cornea treatment with PEDF and DHA also stimulated the synthesis of the docosanoid NPD1. However, the synthetized amount is much lower than RvD6i (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ,
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). When adding NPD1 to injured corneas, there is an increase in the gene expression and protein levels of the neurotrophins NGF, brain-derived neurotrophic factor (BDNF), and semaphorin 7A (Sema7A) that stimulate axon growth (
      • Pham T.L.
      • He J.
      • Kakazu A.H.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.
      ). These proteins are secreted into tears and activate receptors in the corneal nerve terminals to facilitate downstream signaling as well as retrograde to the neurons of the TG.
      Using RNA-sequencing to analyze the gene expression in TG from the injured corneas of mice, we reveal that the product of PEDF+DHA, RvD6i, applied topically to the cornea induces the expression of two interesting genes in the TG, chromosome 9 open reading frame 72 (C9orf72) and glycoprotein M6A (Gpm6A) (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ). These genes stimulate neurogenesis and growth cone formation (
      • Sivadasan R.
      • Hornburg D.
      • Drepper C.
      • Frank N.
      • Jablonka S.
      • Hansel A.
      • Lojewski X.
      • Sterneckert J.
      • Hermann A.
      • Shaw P.J.
      • et al.
      C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.
      ,
      • Formoso K.
      • Garcia M.D.
      • Frasch A.C.
      • Scorticati C.
      Evidence for a role of glycoprotein M6a in dendritic spine formation and synaptogenesis.
      ).
      Ocular pathologies that damage corneal nerves in many cases produce neuropathic pain (
      • Goyal S.
      • Hamrah P.
      Understanding neuropathic corneal pain–gaps and current therapeutic approaches.
      ). In addition, there are a significant number of patients who have symptoms of DED and experience neuropathic pain, suggesting that there is an active cornea-TG relationship (
      • Ferrari G.
      • Bignami F.
      • Giacomini C.
      • Capitolo E.
      • Comi G.
      • Chaabane L.
      • Rama P.
      Ocular surface injury induces inflammation in the brain: in vivo and ex vivo evidence of a corneal–trigeminal axis.
      ). Two genes involved in pain were decreased in corneas treated with RvD6i: Tac1 that encodes substance P (SP), which is one of the most abundant neuropeptides expressed in corneal nerves (
      • Müller L.J.
      • Marfurt C.F.
      • Kruse F.
      • Tervo T.M.T.
      Corneal nerves: structure, contents and function.
      ,
      • He J.
      • Bazan H.E.P.
      Neuroanatomy and neurochemistry of mouse cornea.
      ,
      • He J.
      • Pham T.L.
      • Bazan H.E.P.
      Mapping the entire nerve architecture of the cat cornea.
      ), and Calcb, which encodes calcitonin gene-related peptide (also abundant in corneal nerves) (
      • Müller L.J.
      • Marfurt C.F.
      • Kruse F.
      • Tervo T.M.T.
      Corneal nerves: structure, contents and function.
      ,
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Recovery of corneal sensitivity, calcitonin gene-related peptide-positive nerves, and increased wound healing induced by pigment epithelial-derived factor plus docosahexaenoic acid after experimental surgery.
      ) (Fig. 5C). Both neuropeptides have important roles in neurogenic inflammation and pain (
      • Zieglgänsberger W.
      Substance P and pain chronicity.
      ,
      • Iyengar S.
      • Ossipov M.H.
      • Johnson K.W.
      The role of calcitonin gene-related peptide in peripheral and central pain mechanisms including migraine.
      ). In addition, corneal treatment with RvD6i increased the gene expression of transient receptor potential melastatin 8 (Trpm8) (Fig. 5D). TRPM8 ion channels are cool sensors that regulate the wetting of the ocular surface and produce an analgesic effect on chronic pain (
      • Belmonte C.
      • Gallar J.
      Cold thermoreceptors, unexpected players in tear production and ocular dryness sensations.
      ,
      • Parra A.
      • Madrid R.
      • Echevarria D.
      • del Olmo S.
      • Morenilla-Palao C.
      • Acosta M.C.
      • Gallar J.
      • Dhaka A.
      • Viana F.
      • Belmonte C.
      Ocular surface wetness is regulated by TRPM8-dependent cold thermoreceptors of the cornea.
      ,
      • Proudfoot C.J.
      • Garry E.M.
      • Cottrell D.F.
      • Rosie R.
      • Anderson H.
      • Robertson D.C.
      • Fleetwood-Walker S.M.
      • Mitchell R.
      Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain.
      ,
      • Liu B.
      • Fan L.
      • Balakrishna S.
      • Sui A.
      • Morris J.B.
      • Jordt S.-E.
      TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain.
      ,
      • Fernández-Peña C.
      • Viana F.
      Targeting TRPM8 for pain relief.
      ). Our prior studies in a mouse model where the nerves had been damaged at the level of the anterior stroma, showed that cornea TRPM8-positive nerve fibers only reach 50% of their normal density after 3 months of injury, suggesting that the decrease in TRPM8 may contribute to DE-like pain (
      • He J.
      • Pham T.L.
      • Kakazu A.H.
      • Bazan H.E.P.
      Remodeling of substance P sensory nerves and transient receptor potential melastatin 8 (TRPM8) cold receptors after corneal experimental surgery.
      ). Therefore, decreased expression of SP and calcitonin gene-related peptide and increased expression of TRPM8 after injury and treatment with RvD6i suggests that the new docosanoid could protect corneas from pain. It also opens avenues of potential therapeutic exploration for ocular surface damage, especially corneal neurotrophic ulcers, because previous studies have shown ocular pain as a side effect of increased corneal nerve regeneration caused by topical treatment with NGF (
      • Lambiase A.
      • Rama P.
      • Bonini S.
      • Caprioglio G.
      • Aloe L.
      Topical treatment with nerve growth factor for corneal neurotrophic ulcers.
      ). Previous studies using RvD1 and RvD5 had shown pain attenuation in a mouse model of tibia bone fracture, while RvD3 and RvD4 had no effect (
      • Zhang L.
      • Terrando N.
      • Xu Z.-Z.
      • Bang S.
      • Jordt S.-E.
      • Maixner W.
      • Serhan C.N.
      • Ji R.-R.
      Distinct analgesic action of DHA and DHA-derived specialized pro-resolvin mediators on post-operative pain after bone fracture in the mice.
      ). These differences could be due to different expression of its receptors. In an osteoporosis mouse model, the precursor of RvDs, 17R-HDHA, decreases pain behavior probably through activation of AXL receptors (
      • Huang J.
      • Burston J.J.
      • Li L.
      • Ashraf S.
      • Mapp P.I.
      • Bennett A.J.
      • Ravipati S.
      • Pousinis P.
      • Barret D.A.
      • Scammell B.E.
      • et al.
      Targeting the D series resolvin receptor system for the treatment of osteoarthritis pain.
      ). Another important finding is that RvD6i is a strong inducer of the gene expression of Rictor in the TG (
      • Pham T.L.
      • Kakazu A.H.
      • He J.
      • Jun B.
      • Bazan N.G.
      • Bazan H.E.P.
      Novel RvD6 stereoisomer induces corneal nerve regeneration and wound healing post-injury by modulating trigeminal transcriptomic signature.
      ) (Fig. 5D). RICTOR is a key component of the mammalian target of rapamacyn-insensitive complex 2 (mTORC2) and plays a role in anti-inflammation and axon growth of sensory neurons after injury (
      • Chen N.
      • Zhou P.
      • Liu X.
      • Li J.
      • Wan Y.
      • Liu S.
      • Wei F.
      Overexpression of Rictor in the injured spinal cord promotes functional recovery in a rat model of spinal cord injury.
      ).
      A summarized scheme of the signaling pathways of docosanoids stimulated by PEDF and DHA is shown in Fig. 6.
      Figure thumbnail gr6
      Fig. 6Schematic model of signaling stimulated by the combination of PEDF+DHA. DHA is rapidly incorporated into membrane phospholipids from corneal epithelium and then released after stimulation by PEDF of the PEDF-R with calcium-independent phospholipase A2 (iPLA2ζ) activity. Free DHA is then the substrate for docosanoids such as NPD1 and the novel RvD6i. These docosanoids are then released into tears and, by autocrine stimulation, to an undefined GPRC receptor(s) that induces the gene and protein expression of neurotrophic factors NGF, BDNF, and semaphorin 7A (Sema7A) that are secreted into tears and enhance axon outgrowth. RvD6i stimulates corneal wound healing, corneal sensation and nerve recovery, and tear secretion. The mechanism involves changes in the TG transcriptome with activation of genes related to neurogenesis and modulation of genes implicated in neuropathic pain. Treatment with PEDF or DHA alone does not activate these pathways, and therefore, there was no increase in cornea nerve regeneration (
      • Cortina M.S.
      • He J.
      • Li N.
      • Bazan N.G.
      • Bazan H.E.P.
      Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.
      ). Gpm6A, glycoprotein M6A; C9orf72, chromosome 9 open reading frame 72; Trpm8, transient receptor potential melastatin 8.

      Conclusions

      Cornea innervation plays a pivotal role in maintaining the homeostasis of the ocular surface and tissue clarity (
      • Shaheen B.S.
      • Bakir M.
      • Jain S.
      Corneal nerves in health and disease.
      ). Damage to corneal nerves produces a decrease in tear production and blinking reflex and can impair epithelial wound healing resulting in loss of transparency and vision (
      • Cruzat A.
      • Witkin D.
      • Baniasadi N.
      • Zheng L.
      • Ciolino J.B.
      • Jurkunas U.V.
      • Chodosh J.
      • Pavan-Langston D.
      • Dana R.
      • Hamrah P.
      Inflammation and the nervous system: the connection in the cornea in patients with infectious keratitis.
      ,
      • He J.
      • Bazan H.E.P.
      Corneal nerve architecture in a donor with unilateral epithelial basement membrane dystrophy.
      ,
      • Pham T.L.
      • Kakazu A.
      • He J.
      • Bazan H.E.P.
      Mouse strains and sexual divergence in corneal innervation and nerve regeneration.
      ,
      • Garcia-Gonzalez M.
      • Cañadas P.
      • Gros-Otero J.
      • Rodriguez-Perez I.
      • Cañones-Zafra R.
      • Kozobolis V.
      • Teus M.A.
      Long-term corneal subbasal nerve plexus regeneration after laser in situ keratomileusis.
      ,
      • He J.
      • Bazan H.E.P.
      Mapping the nerve architecture of diabetic human corneas.
      ,
      • Hamrah P.
      • Cruzat A.
      • Dastjerdi M.H.
      • Zheng L.
      • Shahatit B.M.
      • Bayhan H.A.
      • Dana R.
      • Pavan-Langston D.
      Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study.
      ). Therefore, better knowledge on corneal nerve function and repair will increase therapeutic strategies for pathologies that affect corneal innervation. DHA-derived docosanoids, such as the new mediator RvD6i, could serve as potential treatment options to reduce cornea-related inflammation. The effect of this lipid in accelerating nerve regeneration and modulating the gene expression of components of neuropathic pain in the TG could provide a new alternative in the treatment of patients with DE following refractive surgery as well as possible cotreatment to several pathologies that decrease corneal nerve density. Prospective human clinical trials will be needed to confirm optimal dosing, modes of administration, efficacy, and safety of these promising new treatments for DE and ocular surface diseases.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author contributions

      T. L. P. and H. E. P. B. contributed to the conception and design of the article and to interpreting the relevant literature. T. L. P. wrote the first draft, and H. E. P. B. reviewed the manuscript.

      Author ORCIDs

      Funding and additional information

      This work was supported by the US National Institutes of Health, National Eye Institute Grant R01 EY019465 (H.E.P.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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