Current vaccines rely on the sequence of Spike (S) protein to induce immunity against the severe acute respiratory coronavirus-2 (SARS-CoV-2) virus. Because of the high mutation rate of the viral S protein, new mutant strains are developed to generate new infectivity profiles. Bioactive lipid mediators (LMs) derived from docosahexaenoic acid (DHA) are synthesized on demand to sustain homeostasis. The purpose of this study was to determine the action of selected LMs in the viral replication of SARS-CoV-2 Omicron BA.5 variant in human lung and nasal epithelial cells. Cells from healthy donors were infected with Omicron BA.5 for one hour and treated with 500 nM Elovanoid (ELV)-N32, ELV-N34, Resolvin D6 isomer (RvD6i), Neuroprotection D1 (NPD1), or vehicle before and after infection. Impedance was recorded to determine cell death by infectivity. Cells were then immunostained for nucleocapsid (N) protein, microtubule-associated protein 1B-light chain 3 (LC3B), and autophagic proteins. N and S RNA were measured to assess the synthesis of viral components. The addition of ELV-N34 or RvD6i decreased the synthesis of N RNA by 76.7% and 96.9%, respectively, in lung primary culture, while NPD1 exerted the same effect in nasal epithelial cells (61.7% reduction). In lung cells, transcription of autophagy-related gene-3 (ATG3) and Sequestosome 1 (SQSTM1/p62), components of the autophagy initiation process, decreased compared to the non-treated infected cells. The results suggest that specific LMs prevent viral autophagy machinery hijacking, leading to a decrease in BA.5 replication. This novel effect of the bioactive LMs as antivirals, regardless of the protein sequence, would potentially complement vaccination and other prevention and treatment therapeutics.

Neurodegenerative diseases induce astrocyte phenotypes that trigger inflammation and cell damage. This cell induction also takes place in post-acute neurological syndrome from SARS-CoV-2 infection. We found that the secretome from Omicron BA.5 infected human nasal epithelial or human lung cells induced the activation of human astrocytes to a reactive pro-inflammatory phenotype as defined by nuclear translocation of NF-kB/p65. Remarkably, the secretome from these cells incubated with Elovanoid (ELV)-N34 or Resolvin D6 (RvD6)-isomer (500nM) did not trigger the formation of reactive astrocytes. One of the factors involved is CXCL1, secreted by Omicron BA.5-infected nasal epithelial cells. So, when CXCL1 was administered intranasally to mice along with Interferon type I, the infiltration of fluorescein indicated a permeabilization of the neurovascular unit. Astrocytes are close to this barrier and contribute to restricting the access of damaging molecules to the brain parenchyma. Together, these results point to a specific way of entry of chemokines and cytokines as part of the secretome from infected cells that may play a role in long COVID brain sequelae.

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.

PURPOSE:To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. METHODS:To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 μL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. RESULTS:Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. CONCLUSIONS:LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Balanced interplay of cell-type specific transcriptomic regulation is critical for nerve regeneration in the central and peripheral nervous system. As a model, we use the cornea, where we recently discovered the RvD6-isomer and its function as a regulator of nerve regeneration, critical for dry eye in aging. Single-RNA-sequencing (snRNASeq) and spatial transcriptomics were used to interrogate cell populations and gene expression in central cornea/limbus from male, 3-month, C57BL/6 wild-type (WT) and injured mice cornea. Isolated nuclei from cornea/limbus of WT mice (N=12) were processed by 10×Genomics pipeline Cellranger. Central mice corneas were damaged with 2mm trephine, and epithelium and anterior stroma removed with corneal rust ring remover (Algerbrush II). Two days post-injury, corneas were excised, formalin-fixed paraffin-embedded, and 5µm sections prepared for Visium CytAssist mediated probe hybridization, library construction, sequencing, data analysis using Spaceranger, data visualization and cell cluster annotation using Loupe Browser. Cell populations were distinguished by t-distributed stochastic neighbor embedding (t-SNE). RNAscope® assay was used to validate two genes, ALDH3A1 and SPARC.

The first total synthesis of a novel neuroprotective Resolvin D6 enantiomer, 4(R),17(R)-Resolvin D6, is described. Resolvin D6 is a lipid mediator derived from docosahexaenoic acid (DHA, C22:6, x-3) with anti-inflammatory properties. Key synthetic features include the use of a MacMillan enantioselective organocatalytic a-oxidation of aldehydes in conjunction with a chiral pool-based approach to generate chiral synthons, a 1,4 enyne unit as a linchpin, as well as a (Z)-selective Wittig coupling to assemble the Resolvin D6 core carbon skeleton. (C) 2022 Elsevier Ltd. All rights reserved.

To provide a complete nerve architecture and main sensory neuropeptide distribution in the chicken cornea.Adult chickens aged 6 months and 4 years were used. The whole cornea was stained with protein gene product (PGP) 9.5 antibody-a pan marker for nerve fibers, calcitonin gene-related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. Relative corneal epithelial nerve fiber densities, including subbasal bundles and superficial terminals, were assessed by computer-assisted analysis.An average of about 76.3 ± 5.7 (n = 8 corneas, 4 M/4F) stromal nerve trunks enter the cornea radially and are evenly distributed around the limbus with no significant difference between male and female chickens. The subbasal nerve bundles do not extend in a given direction and, as a result, do not form a vortex in the center of the cornea. Furthermore, the chicken cornea contains more SP-positive nerves than CGRP-positive nerves. It is also shown that aging significantly reduces corneal epithelial nerve density in chickens.This is the first study to provide a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the adult chicken cornea. The findings show chicken corneal innervation has many differences to human and mammal cornea.

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.

Innervation sustains cornea integrity. Pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15- or 5-LOX inhibitors, suggesting that the 4- and 17-hydroxy of DHA have an RR- or SR-configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si(1-4) from tears were resolved. The RR-RvD6 standard eluted as a single peak with RvD6(1) while pure SR-RvD6 eluted with RvD6(3). The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that RR-RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. RR-RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic collapse around the world. Effective treatments to mitigate this viral infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells (HCEC) in culture challenged with IFNγ as models of the eye surface to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein to angiotensin-converting enzyme 2 (ACE2). We found that the lipid mediators, elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i) decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ-stimulated HCEC. There was also a concomitant decrease in the binding of Spike RBD with the lipid treatments. Using RNA-seq analysis, we uncovered that the lipid mediators also attenuated the expression of pro-inflammatoy cytokines participating in hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to open therapeutic avenues to counteract virus attachment and entrance to the body.

Purpose: To characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution. Methods: Sprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis. Results: Thick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% +/- 3.5% and 34.9% +/- 5.1%, respectively, of the total nerves. VIP was 18.4% +/- 5.7%, and NPY and TH positive fibers took up 6.92% +/- 2.66% and 2.92% +/- 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks. Conclusion: This study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.

The high-density corneal innervation plays a pivotal role in sustaining the integrity of the ocular surface. We have previously demonstrated that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) promotes corneal nerve regeneration; here, we report the mechanism involved and the discovery of a stereospecific Resolvin D6-isomer (RvD6si) that drives the process. RvD6si promotes corneal wound healing and functional recovery by restoring corneal innervation after injury. RvD6si applied to the eye surface elicits a specific transcriptome signature in the trigeminal ganglion (TG) that includes Rictor , the rapamycin-insensitive complex-2 of mTOR (mTORC2), and genes involved in axon growth, whereas genes related to neuropathic pain are decreased. As a result, attenuation of ocular neuropathic pain and dry eye will take place. Thus, RvD6si opens up new therapeutic avenues for pathologies that affect corneal innervation.

In vivo confocal microscopy (IVCM) allows the evaluation of the living human cornea at the cellular level. The non-invasive nature of this technique longitudinal, repeated examinations of the same tissue over time. Image analysis of two-dimensional time-lapse sequences of presumed immune cells with and without visible dendrites at the corneal sub-basal nerve plexus in the eyes of healthy individuals was performed. We demonstrated evidence that cells without visible dendrites are highly dynamic and move rapidly in the axial directions. A number of dynamic cells were observed and measured from three eyes of different individuals. The total average displacement and trajectory speeds of three cells without visible dendrites (N = 9) was calculated to be 1.12 ± 0.21 and 1.35 ± 0.17 μm per minute, respectively. One cell with visible dendrites per cornea was also analysed. Tracking dendritic cell dynamics in vivo has the potential to significantly advance the understanding of the human immune adaptive and innate systems. The ability to observe and quantify migration rates of immune cells in vivo is likely to reveal previously unknown insights into corneal and general pathophysiology and may serve as an effective indicator of cellular responses to intervention therapies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic tall around the world. Effective treatments to mitigate this virus infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells in culture challenged with IFNγ to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). Elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i), among the lipid mediators studied, consistently decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ stimulated human corneal epithelial cells (HCEC). There was also a concomitant decrease in the binding of spike RBD with the lipid treatments. Concurrently, we uncovered that the lipid mediators also attenuated the expression of cytokines that participate in the cytokine storm, hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to opening therapeutic avenues for COVID-19 by counteracting virus attachment and entrance to the eye and other cells and the ensuing disruptions of homeostasis.

OBJECTIVE To provide a complete nerve architecture and neuropeptide distribution in the cat cornea. ANIMALS STUDIED Two adult domestic cats. PROCEDURE The cat corneas were stained with protein gene product (PGP) 9.5 antibody-a pan marker for nerve fibers-and then divided into four quarters and double labeled with calcitonin gene-related peptide (CGRP) or substance P (SP) antibodies. Relative corneal nerve fiber densities and nerve terminals were evaluated in whole mount images by computer-assisted analysis. RESULTS An average of 21.5 ± 2.1 thick stromal nerves enters the cornea around the limbus where they split into many branches going up to the anterior stroma. Some branches link to each other, but most of them penetrate the basement membrane in the periphery to give origin to subbasal bundles, which run centripetally and merge to form a whirl-like structure (vortex) at the center. These nerve bundles send out many fine terminals that innervate the epithelial cells. Subbasal nerve density and nerve terminals were greater in the center than in the periphery of the cornea. Additionally, CGRP-positive central epithelial nerve fibers and terminals were more abundant than SP-positive nerves and terminals. CONCLUSION The architecture of cat corneal nerves shows similarities to human and mouse cornea innervation. This study provides useful data for researchers who use the cat model to assess corneal nerve pathological alterations, as well as in the veterinary field where corneal opacities, ulcerations, and infections damage the nerves and decrease sensitivity.