Niyazi Acar

Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry.

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms (C24-C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC-ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24-C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.

Time course of ocular surface and lacrimal gland changes in a new scopolamine-induced dry eye model.

BackgroundThe aim of this study was to set up an animal model of dry eye showing disturbance in several components of the lacrimal functional unit, and to describe the time course of the appearance of clinical signs and inflammatory markers.MethodsDry eye was induced in 6-week-old female Lewis rats by a systemic and continuous delivery of scopolamine via osmotic pumps implanted subcutaneously. We first determined the appropriate dose of scopolamine (6, 12.5, or 25xa0mg/day) for 28xa0days. In a second set of experiments, we determined markers after 1, 2, 3, 7, 10, 17, or 28xa0days of a 12.5-mg/day dose. Clinical signs of corneal dryness were evaluated in vivo using fluorescein staining. MHC II expression and mucin Muc5AC production were detected on the conjunctival epithelium using immunostaining. The level of IL-1β, IL-6, TNF-α, and IFN-γ mRNA was evaluated by real-time polymerase chain reaction in conjunctiva and exorbital lacrimal gland (LG). Lipids were extracted from the exorbital LG for fatty acid analysis.ResultsDaily scopolamine doses of 12.5xa0mg and 25xa0mg applied for a 28-day period induced keratitis, a decrease in Muc5AC immunostaining density in the conjunctival epithelium, and modifications in the fatty acid composition of the exorbital LG. Animals treated with a 12.5-mg/day dose of scopolamine exhibited an increase in corneal fluorescein staining after 2, 10, and 28xa0days. All animals exhibited unilateral or bilateral keratitis after 17xa0days. In the conjunctival epithelium, a significant decrease in Muc5AC immunostaining density was observed at early and late time points, and MHC II expression tended to be increased after 1, 7, 10, and 28xa0days, without reaching statistical significance. The levels of TNF-α, IL-1β and IL-6xa0mRNA were increased with scopolamine treatment in both conjunctiva and exorbital LG. Arachidonic acid and the Δ5 desaturase index were significantly increased in the exorbital LG of dry eye animals at each time point.ConclusionsThis systemic and continuous scopolamine-induced model of dry eye in the rat may represent a helpful tool to investigate moderate dry eye, and makes a contribution in the field of dry eye study.

Nutrition for the Eye: Different Susceptibility of the Retina and the Lacrimal Gland to Dietary Omega-6 and Omega-3 Polyunsaturated Fatty Acid Incorporation

The purpose of this study was to compare the susceptibility of the retina and the exorbital lacrimal gland to dietary supplies of long-chain omega-3 (ω3) and omega-6 (ω6) polyunsaturated fatty acids (LC-PUFAs). Male Wistar rats were fed a 5% lipid diet containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA or (4) a balanced diet deprived of EPA, DHA and GLA for 3 months. Lipids were extracted from plasma phospholipids, retina and exorbital lacrimal gland, and fatty acid composition was determined by gas chromatography. Dietary supplementation with EPA and DHA increased ω3 PUFA levels in plasma phospholipids as well as in the retina and the exorbital lacrimal gland. By contrast, GLA supplementation favored ω6 PUFA incorporation, and particularly the incorporation of the end-chain ω6 product, docosapentaenoic acid (DPA), into all tissues. Supplementation with EPA, DHA and GLA increased the levels of DHA, EPA and dihomo-GLA (dGLA), whereas arachidonic acid (AA) was unchanged and DPA decreased in the retina and the lacrimal gland. The ability of both tissues to incorporate PUFAs from blood was evaluated. The results showed that the retina was more selective than the lacrimal gland for EPA. In spite of the different susceptibility of the retina and the lacrimal gland to dietary PUFAs, these results suggest that the concomitant use of dietary ω3 and ω6 PUFAs may be useful in modulating inflammation in both tissues.

Retinas of the diurnal rodent Arvicanthis ansorgei are highly resistant to experimentally induced stress and degeneration.

PURPOSEnEnvironmentally induced stress plays a significant role in retinal degeneration and blindness both in animals and in humans. Among such sources of stress, phototoxicity is well studied and has been shown to lead to photoreceptor-specific loss in a number of species. However, the vast majority of studies have been conducted in nocturnal, albino rod-dominant rat and mouse strains, and the pertinence of such findings to human pathology and cone loss is debatable. The authors examined retinal vulnerability to damage in the diurnal murid rodent Arvicanthis ansorgei, a pigmented species with a large number of cones.nnnMETHODSnThe authors used established protocols for exposing animals to a wide range of lighting conditions (variable intensity, duration, spectrum, previous light history, and time of exposure) and injecting N-methyl-N-nitrosourea (MNU); each procedure is reported to produce rapid and complete photoreceptor-specific damage. Animals then underwent electroretinography to record rod and cone function and were subsequently euthanized and used for immunohistochemical analysis of retinal structure and quantification of free fatty acids.nnnRESULTSnThese standard regimens produced no detectable detrimental effects on A. ansorgei retinal phenotype, function, or structure. Partial retinal damage in A. ansorgei was induced by very intense blue light or elevated doses of MNU. This resistance was not attributable to differences in lipid composition (specifically, docosahexaenoic acid) between A. ansorgei and susceptible strains of mice and rats.nnnCONCLUSIONSnThe retina of this species exhibits exceptionally high resistance to damage from light and toxins such as MNU.

Decreasing dietary linoleic acid promotes long chain omega-3 fatty acid incorporation into rat retina and modifies gene expression.

Age-related macular degeneration (AMD) may be partially prevented by dietary habits privileging the consumption of ω3 long chain polyunsaturated fatty acids (ω3s) while lowering linoleic acid (LA) intake. The present study aimed to document whether following these epidemiological guidelines would enrich the neurosensory retina and RPE with ω3s and modulate gene expression in the neurosensory retina. Rat progenitors and pups were fed with diets containing low or high LA, and low or high ω3s. After scotopic single flash and 8-Hz-Flicker electroretinography, rat pups were euthanized at adulthood. The fatty acid profile of the neurosensory retina, RPE, liver, adipose tissue and plasma was analyzed using gas chromatography. Gene expression was analyzed with real-time PCR in the neurosensory retina. Diets rich in ω3s efficiently improved the incorporation of ω3s into the organs and tissues. This raising effect was magnified by lowering LA intake. Compared to a diet with high LA and low ω3s, low LA diets significantly upregulated LDL-receptor gene expression. Similar but not significant upregulation of CD36, ABCA1, ALOX5 and ALOX12 gene expression was observed in rats fed with low LA. No effect was observed on retinal function. Increasing the intake in ω3s and lowering LA improved the enrichment with ω3s of the tissues, including the neurosensory retina and RPE, and upregulated genes involved in lipid trafficking in the neurosensory retina. Those results consistently reinforced the beneficial role of ω3s in the prevention of AMD, especially when the diet contained low levels of LA, as suggested from epidemiological data.

24S-hydroxycholesterol and cholesterol-24S-hydroxylase (CYP46A1) in the retina: from cholesterol homeostasis to pathophysiology of glaucoma

Free cholesterol is the predominant form of cholesterol in the neural retina. The vertebrate neural retina exhibits its own capacity to synthesize cholesterol and meets its demand also by taking it from the circulation. Defects in cholesterol synthesis and trafficking in the neural retina has detrimental consequences on its structure and function, highlighting the crucial importance of maintaining cholesterol homeostasis in the retina. Our purpose was to give a review on the functioning of the retina, the role of cholesterol and cholesterol metabolism therein, with special emphasis on cholesterol-24S-hydroxylase (CYP46A1). Similar to the brain, the retina expresses cholesterol-24S-hydroxylase (CYP46A1) and is enriched in its metabolic product, 24S-hydroxycholesterol. We recently published that one single nucleotide polymorphism in CYP46A1 gene, designated as rs754203, was a risk factor for glaucoma. Glaucoma is the second leading cause of blindness worldwide, affecting more than 60 million people. Glaucoma is characterized by the loss of retinal ganglion cells, which show high CYP46A1 expression. These data suggest the potential involvement of CYP46A1 and 24S-hydroxycholesterol in the pathophysiology of glaucoma.

Modulation of brain PUFA content in different experimental models of mice

The relative amounts of arachidonic acid (AA) and docosahexaenoic acid (DHA) govern the different functions of the brain. Their brain levels depend on structures considered, on fatty acid dietary supply and the age of animals. To have a better overview of the different models available in the literature we here compared the brain fatty acid composition in various mice models (C57BL/6J, CD1, Fat-1, SAMP8 mice) fed with different n-3 PUFA diets (deficient, balanced, enriched) in adults and aged animals. Our results demonstrated that brain AA and DHA content is 1) structure-dependent; 2) strain-specific; 3) differently affected by dietary approaches when compared to genetic model of PUFA modulation; 4) different in n-3 PUFA deficient aged C57BL6/J when compared to SAMP8 mouse model of aging. From these experiments, we highlight the difficulty to compare results obtained in different mouse models, different strains, different brain regions and different ages.

Ocular surface assessment in soft contact lens wearers; the contribution of tear osmolarity among other tests

Purpose: To determine whether tear osmolarity contributes to the assessment of the ocular surface in soft contact lens (CL) wearers.

Polyunsaturated fatty acids induce modification in the lipid composition and the prostaglandin production of the conjunctival epithelium cells.

BackgroundThis study was conducted to evaluate whether polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA), as found in the diet, may affect the lipid composition of conjunctival epithelium and whether these modifications affect prostaglandin (PG) production after inflammatory stimulation.MethodsChang and IOBA-NHC conjunctival human cells were treated with GLA and/or EPA at 5, 10, 20, 30, 40, or 50xa0μg/ml for 72xa0h and then were stimulated with interferon-gamma (IFN-γ) for 48xa0h. Changes in the composition of neutral lipids and phospholipids were monitored by gas chromatography. PGE1 and PGE2 levels were measured by enzyme immunoassay.ResultsPUFA supplementations in the culture medium induced incorporation of these fatty acids and of their metabolites in neutral lipids and phospholipids of the conjunctival cells. The fatty acid composition of neutral lipids and phospholipids was not affected by stimulation with IFN-γ. The production of PGE1 and PGE2 was affected by GLA supplementation whereas it was not modified by EPA supplementation. A combined supplementation of EPA and GLA did not change the production of PGE1 but decreased the production of PGE2.ConclusionsThese results suggest that modulation of fatty acid composition and PG production by PUFA supplementation is possible in the conjunctival epithelium, which is an important site of inflammation in dry eye syndrome.

Pantethine Alters Lipid Composition and Cholesterol Content of Membrane Rafts, With Down-Regulation of CXCL12-Induced T Cell Migration

Pantethine, a natural low‐molecular‐weight thiol, shows a broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We have recently shown that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinsons disease through multiple mechanisms. In the present study, we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to a chronic inflammation. We found that pantethine‐treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12‐driven chemotaxis, and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: (i) CXCL12 binding to its target cells; (ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; and (iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which had their palmitoylation level reduced.. In conclusion, the results presented here, together with previously published findings, indicate that due to its pleiotropic action, pantethine can downregulate the multifaceted process leading to pathogenic T cell activation and migration. J. Cell. Physiol. 230: 2415–2425, 2015.