Omega-3 Polyunsaturated Fatty Acids and Their Anti-Oxidant, Anti-Inflammatory and Neuroprotective Effects in Diabetic Retinopathy: A Narrative Review.

Diabetic retinopathy (DR) is a common microvascular complication of type 2 Diabetes Mellitus (T2DM) that can have vision-threatening consequences, particularly if it advances to the proliferative stage and is left untreated. Owing to the central role of hyperglycemia-induced oxidative stress, multiple anti-oxidants have been investigated for their therapeutic value. However, there is a lack of substantial data to support the use of any of the compounds tested so far. Omega-3 polyunsaturated fatty acids (PUFAs), namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have received much acclaim for their positive impact on cardiovascular health outcomes. The anti-oxidative, anti-inflammatory and neuroprotective properties of PUFAs also make them promising therapeutic and preventive agents for DR. The current evidence is derived mainly from in vitro and animal studies and provides some insight into the underlying mechanisms involved. These fatty acids are capable of direct anti-oxidative and anti-inflammatory effects. They also concomitantly promote intrinsic defense mechanisms and recovery, particularly of photoreceptor neurons. Hence, dietary supplementation with PUFAs, mainly from marine sources, can halt and reverse the retinal damage seen in DR. Furthermore, clinical trials have reported improved vision and quality of life in DR patients after supplementation. However, a major limitation of these trials is the use of nutraceutical formulations in which omega-3 PUFAs are combined with other anti-oxidant compounds, thereby preventing the evaluation of omega-3 as standalone treatment. Although the results of experimental studies to date have been promising, more clinical trials are required to determine the full extent of benefits in patients with DR.

Diabetic retinopathy and the role of Omega-3 PUFAs: A narrative review.

Diabetes mellitus has been a major cause of concern for the past few decades. As the number of diabetic patients increases, so too does the occurrence of its complications. Diabetic retinopathy (DR) is one of these and constitutes the most common cause of blindness amongst working-age individuals. Chronic exposure to a hyperglycaemic environment remains the driving force of a cascade of molecular events that disrupt the microvasculature of the retina and if left untreated can lead to blindness. In this review, we identify oxidative stress as a major implication in the pathway to the development of DR and speculate that it plays a central role especially in the early stages of the disease. Cells lose their antioxidant capacity under a hyperglycaemic state, free radicals are formed and eventually apoptosis ensues. The polyol pathway; advanced glycation end-product formation; the protein kinase C pathway, and the hexosamine pathway are found to contribute to the increase in oxidative stress observed in diabetic patients. We also investigate the use of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in DR. These molecules possess antioxidant and anti-inflammatory properties and have been previously investigated for use in other ocular pathologies with promising results. In this review we present the latest findings in pre-clinical and clinical studies for the use of ω-3 PUFAs in DR. We hypothesise that ω-3 PUFAs could be beneficial for DR in ways of reducing the oxidative stress and limiting the progression of the disease that threatens the eyesight of the patient, in conjunction with conventional therapy.

Omega-3 fatty acids promote neuroprotection, decreased apoptosis and reduced glial cell activation in the retina of a mouse model of OPA1-related autosomal dominant optic atrophy.

The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation in a mouse model of OPA1-associated autosomal dominant optic atrophy (ADOA). The blood level of arachidonic acid (AA) and eicosapentaenoic acid (EPA) served to adjust the treatment dosage (AA/EPA = 1.0-1.5). Eight-month-old mice were allocated to four groups (n = 20/group): the ω3-PUFA-treated Opa1enu/+, untreated Opa1enu/+, ω3-PUFA-treated wild-type and untreated wild-type groups. Treated mice received the ω3-PUFAs, EPA and docosahexaenoic acid (DHA; 5:1 ratio) by daily gavage for 4 months based on the measured AA/EPA ratio. Blood, retina and optic nerve (ON) fatty acid levels were determined by gas chromatography, and the retina and ON were histologically examined. Western blotting and/or immunohistochemistry was performed to analyse retinal mediators involved in Opa1-mutation-mediated apoptosis, inflammation and oxidative stress. Increased EPA and reduced AA levels were primarily observed predominantly in the blood and retinal tissues, and a similarly high EPA level tended to be observed in the ONs of ω3-PUFA-treated mice. Retinal ganglion cell and ON axonal densities were higher in both mouse strains upon ω3-PUFA treatment than in the corresponding untreated groups. Caspase-3 expression analysis showed fewer apoptotic retinal cells in both groups of treated mice. Decreases in inflammatory microglia and astrocytes activation and proapoptotic Bcl-2-associated X protein (Bax) expression were noted in the treated groups, with no difference in the antioxidant superoxide dismutase-2 expression. ω3-PUFA supplementation had neuroprotective effects on the retinas of Opa1enu/+ and wild-type mice via blockade of microglia and astrocytes activation and suppression of Bax and caspase-3. Our findings indicated that inhibition of oxidative stress may not be involved in ω3-PUFA-mediated neuroprotection. These novel findings support the use of ω3-PUFAs as a beneficial therapy in the occurrence of ADOA, posing the basis for future clinical trials to confirm these observations.

Omega-3 fatty acids supplementation protects the retina from age-associated degeneration in aged C57BL/6J mice.

OBJECTIVE: To evaluate the therapeutic effects of omega-3 (ω3) fatty acids in the retina of aged mice when the blood arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is maintained between 1.0 and 1.5. METHODS AND ANALYSIS: Aged (24-month-old) wild-type C57BL/6J mice were allocated to two groups: ω3 treated and untreated. Treatment with ω3 was by daily gavage administration of EPA and docosahexaenoic acid for 60 days. Gas chromatography was used to identify and quantify fatty acids in the blood and retina. To count lipofuscin granules and measure the photoreceptor layer, eyecups were examined histologically using transmission electron microscopy and light microscopy. We also analysed eyecups using mass spectrometry-based proteomics. RESULTS: AA levels were lower, and EPA levels were higher, in the blood and retinas of the ω3-treated group than in the untreated group, resulting in a lower AA/EPA ratio. The ω3-treated group also showed significantly fewer lipofuscin granules and a thicker outer nuclear layer than the untreated group. Proteomic analysis revealed significantly greater expression of myelin basic protein, myelin regulatory factor-like protein, myelin proteolipid protein and glial fibrillar acidic protein in the ω3-treated group than in the untreated group. Three different pathways were significantly affected by ω3 treatment: fatty acid elongation, biosynthesis of unsaturated fatty acids and metabolic pathways. CONCLUSION: Two months of ω3 supplementation (when the blood AA/EPA~1.0-1.5) in aged mice reduced lipofuscin granule formation in the retina and protected the photoreceptor layer, suggesting that ω3 supplementation slows normal age-related retinal degeneration.

Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease.

Purpose: To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5. Methods: Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins. Results: EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 µm) than in the wild-type (61.40 ± 1.84 µm) or untreated ABCA4-/- (56.50 ± 3.24 µm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response. Conclusions: Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.

Omega-3 fatty acids protect retinal neurons in the DBA/2J hereditary glaucoma mouse model.

The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation, alone or in combination with timolol eye drops, in a mouse model of hereditary glaucoma. DBA/2J mice (8.5-month-old) were assigned to an ω3-PUFAs + timolol, ω3-PUFAs only, timolol only, or an untreated group. Treated mice received a daily gavage administration of eicosapentaenoic acid (EPA) and docosahexaenoic acid and/or topical instillation of timolol (0.5%) once a day for 3 months. Blood was analysed regularly to determine ω3-PUFA levels and retinas were histologically analysed. Real-time PCR and Western blot were performed for retinal pro-inflammatory cytokines and macrophages. Blood arachidonic acid/EPA ratio gradually decreased and reached the desired therapeutic range (1-1.5) after 4 weeks of daily gavage with ω3-PUFAs in the ω3-PUFAs + timolol and ω3-PUFAs only groups. Retinal ganglion cell densities were significantly higher in the ω3-PUFAs + timolol (1303.77 ± 139.62/mm2), ω3-PUFAs only (768.40 ±â€¯52.44/mm2) and timolol only (910.57 ±â€¯57.28/mm2) groups than in the untreated group (323.39 ±â€¯95.18/mm2). ω3-PUFA supplementation alone or timolol alone, significantly increased protein expression levels of M1 macrophage-secreted inducible nitric oxide synthase and M2 macrophage-secreted arginase-1 in the retina, which led to significant decreases in the expression levels of tumour necrosis factor-α (TNF-α). ω3-PUFA supplementation alone also resulted in significantly reduced expression of interleukin-18 (IL-18). ω3-PUFA + timolol treatment had no effect on the expression level of any of the aforementioned mediators in the retina. Supplementation with ω3-PUFAs has neuroprotective effect in the retinas of DBA/2J mice that is enhanced when combined with timolol eye drops. The continued inflammation following ω3-PUFAs + timolol treatment suggests that downregulation of IL-18 and TNF-α may not be the only factors involved in ω3-PUFA-mediated neuroprotection in the retina.

Neuroprotective Effects of Omega-3 Polyunsaturated Fatty Acids in a Rat Model of Anterior Ischemic Optic Neuropathy.

Purpose: The purpose of this study was to investigate the therapeutic effect of omega-3 polyunsaturated fatty acid (ω-3 PUFA) administration in a rat model of anterior ischemic optic neuropathy (rAION). Methods: The level of blood arachidonic acid/eicosapentaenoic acid (AA/EPA) was measured to determine the suggested dosage. The rAION-induced rats were administered fish oil (1 g/day EPA) or phosphate-buffered saline (PBS) by daily gavage for 10 consecutive days to evaluate the neuroprotective effects. Results: Blood fatty acid analysis showed that the AA/EPA ratio was reduced from 17.6 to ≤1.5 after 10 days of fish oil treatment. The retinal ganglion cell (RGC) densities and the P1-N2 amplitude of flash visual-evoked potentials (FVEP) were significantly higher in the ω-3 PUFA-treated group, compared with the PBS-treated group (P < 0.05). The number of apoptotic cells in the RGC layer of the ω-3 PUFA-treated rats was significantly decreased (P < 0.05) compared with that of the PBS-treated rats. Treatment with ω-3 PUFAs reduced the macrophage recruitment at the optic nerve (ON) by 3.17-fold in the rAION model. The M2 macrophage markers, which decrease inflammation, were induced in the ω-3 PUFA-treated group in contrast to the PBS-treated group. In addition, the mRNA levels of tumor necrosis factor-alpha, interleukin-1 beta, and inducible nitric oxide synthase were significantly reduced in the ω-3 PUFA-treated group. Conclusions: The administration of ω-3 PUFAs has neuroprotective effects in rAION, possibly through dual actions of the antiapoptosis of RGCs and anti-inflammation via decreasing inflammatory cell infiltration, as well as the regulation of macrophage polarization to decrease the cytokine-induced injury of the ON.

Therapeutic potential of omega-3 fatty acids supplementation in a mouse model of dry macular degeneration.

Purpose: To evaluate the therapeutic effects of omega-3 (ω-3) and omega-6 (ω-6) fatty acids in the CCL2-/- model of dry age-related macular degeneration (AMD). The blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA) served to adjust the treatment dosage (AA/EPA=1-1.5). Methods: Nine-month-old animals were allocated to different groups: (A) C57BL/6 untreated , (B) CCL2-/- untreated, (C) CCL2-/- treated with ω-3+ω-6, and (D) CCL2-/- treated with ω-3. Treatment was daily administered by gavage for 3 months. Fatty acids analysis was performed and retinas were histologically examined. Three-month-old wild type mice were used for comparison purposes. Real-time PCR and Western blot were performed for retinal inflammatory mediators. Results: Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment groups. The outer nuclear layer thickness was increased in groups C (90.0±7.8 µm) and D (125.6±9.8 µm) [corrected] compared with groups B (65.6±3.0 µm) and A (71.1±4.2 µm), and in young mice, it was 98.0±3.9 µm. A decrease in NF-κB expression was noted in the treatment groups. Interleukin (IL) 18 protein levels demonstrated a significant reduction in the ω-3-treated group only. Conclusion: Supplementation with ω-3+ω-6 or ω-3 alone (AA/EPA=1-1.5) suggests a protective mechanism in the CCL2-/- animal model of dry AMD, with a more beneficial effect when ω-3 are used alone. Our findings indicated that inflammation is not the only determining factor; perhaps a regenerative process might be involved following administration of ω-3 fatty acids.

The New Era of Omega-3 Fatty Acids Supplementation: Therapeutic Effects on Dry Age-Related Macular Degeneration.

One of the major causes of reduced vision over the age of 50 is age-related macular degeneration (AMD). Although such a common pathology, there are no current guidelines for the first-line treatment of dry AMD. The aim of this study is to evaluate the therapeutic effects of high omega-3 fatty acids as anti-inflammatory agents in two sub-groups of dry AMD patients with 1) mild to moderate visual impairment and 2) with severe visual impairment (blindness). The key feature of this investigation is the frequent monitoring of the levels of specific fatty acids in patient's blood in order to adjust the treatment dose within the ideal therapeutic window. Following the positive outcome from our initial observational studies in patients with dry AMD, who demonstrated significant improvement in visual acuity (gain of ≥ 1 line of vision in 4.5 months) when taking a total of 5 g/day eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), additional studies were encouraged. The latest data which is presented in this chapter suggests that the eyes which had the greatest gain in vision (≥ 15 letters gain at 6 months) were from patients with mild to moderate visual impairment, who were taking between 5-7.5 g/day EPA and DHA and had a ratio of arachidonic acid (AA)/EPA < 2. In addition, a sub-group of dry AMD patients with severe visual impairment (< 6/60), showed significant increase in their visual acuity only 3 months following treatment with omega-3 fatty acids. The preliminary results indicate a promising therapeutic regime for dry AMD and perhaps for other types of retinopathies as well. Although initial results are encouraging, further investigations are necessary to establish a better understanding of the mode of action of these supplements and to observe their long-term effects.

Potentiation of the activity of cisplatin in a human colon tumour xenograft model by auristatin PYE, a structural modification of dolastatin 10.

Dolastatin 10, a marine natural product peptide, is now known to act as a vascular disrupting agent (VDA). These VDA properties were not known when other aspects of its promising pre-clinical profile led to initial unsuccessful clinical trials. Auristatin PYE, a synthetic analogue of dolastatin 10, has demonstrated improved activity in preliminary in vivo studies. However, as with other VDAs, tumour eradication was incomplete due to the maintenance of functional vasculature supporting the viable tumour at the periphery of the tumour xenograft, meaning that once the VDA effect subsides, the tumour regrows. One possible strategy for removing this peripheral tumour involves combining VDA therapy with another anticancer drug with a different mechanism of action. Here, we evaluated the effect of combining auristatin PYE with cisplatin in an HCT-116 human colon adenocarcinoma xenograft model. The effects on the growth of subcutaneously implanted HCT-116 xenografts in mice following intraperitoneal administration of a single dose of 4 mgkg-1 cisplatin and intravenous administration of 1 mgkg-1 auristatin PYE were evaluated compared to the effect of each agent administered alone. The effects on the functional tumour vasculature were also assessed. Statistically significant potentiation (p<0.01) was noted with a 465% growth delay for the combination group compared to the control, and 142 and 310% growth delays for the cisplatin and auristatin PYE groups, respectively. Shut down of tumour vasculature in the combination group was similar to that observed with auristatin PYE on its own. Auristatin PYE demonstrated synergistic antitumour effects when combined with cisplatin, suggesting that a combination chemotherapy regimen would be the most effective strategy when applying this new anticancer drug.