Antioxidant properties of amniotic membrane: novel observations from a pilot study



      Amniotic membrane (AM) is used to manage various debilitated ocular surface conditions. The impact of oxidative stress and free radicals on the ocular surface is increasingly being recognized. Hyaluronic acid (HA) has anti-inflammatory properties and is abundantly present in AM. In this in vitro pilot study, we investigated the potential of AM for intrinsic free radical scavenging properties.


      Strips of AM were incubated in sealed tubes with hydrogen peroxide (H2O2). After being sonicated, uptake of reactive oxygen species (ROS) was measured by the Amplex Red Hydrogen Peroxide/Peroxidase assay. For comparison, 1630 kDA HA was used.


      There was uptake of ROS by all AM samples, which decreased with increasing concentrations of H2O2. Mean ROS uptake for 5 different AMs at 1 hour was significantly greater for 50 μM (83%; SD 11.7, SEM 5.23) compared with 100 μM (67%; SD 20.48, SEM 9.16; p = 0.028; 95% CI 2.8–29.2). The HA comparison group showed similar uptake and trend.


      This pilot study demonstrates that AM is able to remove ROS from its environment. Demonstrating total antioxidant capacity in AM provides evidence for use as a free radical scavenger. The antioxidant properties of AM and the contribution from HA require more research.



      La membrane amniotique (MA) sert à gérer divers états de débilitation des surfaces oculaires. La reconnaissance de l’impact du stress oxydant et des radicaux libres sur la surface oculaire augmente. L’acide hyaluronique (AH) a des propriétés anti-inflammatoires et abonde dans la MA. Dans cette étude pilote in vitro, nous avons examiné le potentiel de la MA concernant ses propriétés anti-radicalaires intrinsèques.


      Étude pilote in vitro.


      Des bandes de MA ont été incubées dans des tubes scellés avec de l’eau oxygénée (H2O2). Après la soumission à l’ultrason, la prise des dérivés réactifs d'oxygène (DRO) a été mesurée par le test Amplex de peroxyde/peroxydase d’hydrogène rouge. L’AH de 1630kDA a servi à la comparaison.


      Il y eut des prises des DRO dans tous les échantillons de MA, qui diminuèrent avec l’augmentation des concentrations de H202. La moyenne des prises des DRO de 5 MA différentes après 1 heure était considérablement plus grande pour 50uM (83 %; SD 11,7, SEM 5,23) comparativement à 100uM (67 %; SD 20,48, SEM 9,16; p = 0,028, 95% CI (2,8, 29,2)). La comparaison du groupe AH a montré une prise et une tendance similaires.


      Cette étude pilote démontre que la MA peut éliminer les DRO de son environnement. La démonstration de l’entière capacité antioxydante de l’AM fournit l’évidence de son utilisation comme puissant anti-radicalaire. Les propriétés d’antioxydant de la MA et la contribution de l’AH requièrent plus de recherches.
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        • Chen Y.
        • Mehta G.
        • Vasiliou V.
        Antioxidant defenses in the ocular surface.
        Ocul Surf. 2009; 7: 176-185
        • Shoham A.
        • Hadziahmetovic M.
        • Dunaief J.L.
        • Mydlarski M.B.
        • Schipper H.M.
        Oxidative stress in diseases of the human cornea.
        Free Radic Biol Med. 2008; 45: 1047-1055
        • Terlecky S.R.
        • Terlecky L.J.
        • Peroxisomes Giordano CR.
        Peroxisomes, oxidative stress, and inflammation.
        World J Biol Chem. 2012; 3: 93-97
        • Saijyothi A.V.
        • Fowjana J.
        • Madhumathi S.
        • et al.
        Tear fluid small molecular antioxidants profiling shows lowered glutathione in keratoconus.
        Exp Eye Res. 2012; 103C: 41-46
        • Azizi B.
        • Ziaei A.
        • Fuchsluger T.
        • Schmedt T.
        • Chen Y.
        • Jurkunas U.V.
        p53-regulated increase in oxidative-stress–induced apoptosis in Fuchs endothelial corneal dystrophy: a native tissue model.
        Invest Ophthalmol Vis Sci. 2011; 52: 9291-9297
        • Matthaei M.
        • Meng H.
        • Meeker A.K.
        • Eberhart C.G.
        • Jun A.S.
        Endothelial Cdkn1a (p21) Overexpression and accelerated senescence in a mouse model of fuchs endothelial corneal dystrophy.
        Invest Ophthalmol Vis Sci. 2012; 53: 6718-6727
        • Macdonald E.C.
        • Cauchi P.A.
        • Azuara-Blanco A.
        • Foot B.
        Surveillance of severe chemical corneal injuries in the UK.
        Br J Ophthalmol. 2009; 93: 1177-1180
        • Fish R.
        • Davidson R.S.
        Management of ocular thermal and chemical injuries, including amniotic membrane therapy.
        Curr Opin Ophthalmol. 2010; 21: 317-321
        • Gicquel J.J.
        Management of ocular surface chemical burns.
        Br J Ophthalmol. 2011; 95: 159-161
        • Shahriari H.A.
        • Tokhmehchi F.
        • Reza M.
        • Hashemi N.F.
        Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model.
        Cornea. 2008; 27: 1148-1150
        • Liang L.
        • Li W.
        • Ling S.
        • Sheha H.
        • Qiu W.
        • Li C.
        • Liu Z.
        Amniotic membrane extraction solution for ocular chemical burns.
        Clin Experiment Ophthalmol. 2009; 37: 855-863
        • Choi J.A.
        • Choi J.S.
        • Joo C.K.
        Effects of amniotic membrane suspension in the rat alkali burn model.
        Mol Vis. 2011; 17: 404-412
        • Rahman I.
        • Said D.G.
        • Maharajan V.S.
        • Dua H.S.
        Amniotic membrane in ophthalmology: indications and limitations.
        Eye (Lond). 2009; 23: 1954-1961
        • Tandon R.
        • Gupta N.
        • Kalaivani M.
        • Sharma N.
        • Titiyal J.S.
        • Vajpayee R.B.
        Amniotic membrane transplantation as an adjunct to medical therapy in acute ocular burns.
        Br J Ophthalmol. 2011; 95: 199-204
        • Tamhane A.
        • Vajpayee R.B.
        • Biswas N.R.
        • et al.
        Evaluation of amniotic membrane transplantation as an adjunct to medical therapy as compared with medical therapy alone in acute ocular burns.
        Ophthalmology. 2005; 112: 1963-1969
        • Joseph A.
        • Dua H.S.
        • King A.J.
        Failure of amniotic membrane transplantation in the treatment of acute ocular burns.
        Br J Ophthalmol. 2001; 85: 1065-1069
        • Takahashi H.
        • Igarashi T.
        • Fujimoto C.
        • Ozaki N.
        • Ishizaki M.
        Immunohistochemical observation of amniotic membrane patching on a corneal alkali burn in vivo.
        Jpn J Ophthalmol. 2007; 51: 3-9
        • He H.
        • Li W.
        • Tseng D.Y.
        • et al.
        Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-alpha-inhibitor (HC*HA) purified from extracts of human amniotic membrane.
        J Biol Chem. 2009; 284: 20136-20146
        • Higa K.
        • Shimmura S.
        • Shimazaki J.
        • Tsubota K.
        Hyaluronic acid-CD44 interaction mediates the adhesion of lymphocytes by amniotic membrane stroma.
        Cornea. 2005; 24: 206-212
        • Wu H.
        • Zhang H.
        • Wang C.
        • et al.
        Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells.
        Mol Vis. 2011; 17: 3364-3370
        • Ye J.
        • Wu H.
        • Wu Y.
        • et al.
        High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells.
        Eye (Lond). 2012; 26: 1012-1020
        • Pauloin T.
        • Dutot M.
        • Warnet J.M.
        • Rat P.
        In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride.
        Eur J Pharm Sci. 2008; 34: 263-273
        • Pauloin T.
        • Dutot M.
        • Joly F.
        • Warnet J.M.
        • Rat P.
        High molecular weight hyaluronan decreases UVB-induced apoptosis and inflammation in human epithelial corneal cells.
        Mol Vis. 2009; 15: 577-583
        • Miki Y.
        • Teramura T.
        • Tomiyama T.
        • et al.
        Hyaluronan reversed proteoglycan synthesis inhibited by mechanical stress: possible involvement of antioxidant effect.
        Inflamm Res. 2010; 59: 471-477
        • Gao F.
        • Koenitzer J.R.
        • Tobolewski J.M.
        • et al.
        Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan.
        J Biol Chem. 2008; 283: 6058-6066
        • Saari H.
        Oxygen derived free radicals and synovial fluid hyaluronate.
        Ann Rheum Dis. 1991; 50: 389-392
        • Saari H.
        • Konttinen Y.T.
        • Friman C.
        • Sorsa T.
        Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate.
        Inflammation. 1993; 17: 403-415
        • Hernnäs J.
        • Nettelbladt O.
        • Bjermer L.
        • Särnstrand B.
        • Malmström A.
        • Hällgren R.
        Alveolar accumulation of fibronectin and hyaluronan precedes bleomycin-induced pulmonary fibrosis in the rat.
        Eur Respir J. 1992; 5: 404-410
        • Hällgren R.
        • Samuelsson T.
        • Laurent T.C.
        • Modig J.
        Accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome.
        Am Rev Respir Dis. 1989; 139: 682-687
        • Casalino-Matsuda S.M.
        • Monzón M.E.
        • Forteza R.M.
        Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium.
        Am J Respir Cell Mol Biol. 2006; 34: 581-591
        • Burlingame J.M.
        • Esfandiari N.
        • Sharma R.K.
        • Mascha E.
        • Falcone T.
        Total antioxidant capacity and reactive oxygen species in amniotic fluid.
        Obstet Gynecol. 2003; 101: 756-761
        • Park W.C.
        • Tseng S.C.
        Modulation of acute inflammation and keratocyte death by suturing, blood, and amniotic membrane in PRK.
        Invest Ophthalmol Vis Sci. 2000; 41: 2906-2914
        • Wang M.X.
        • Gray T.B.
        • Park W.C.
        • et al.
        Reduction in corneal haze and apoptosis by amniotic membrane matrix in excimer laser photoablation in rabbits.
        J Cataract Refract Surg. 2001; 27: 310-319
        • Heiligenhaus A.
        • Bauer D.
        • Meller D.
        • Steuhl K.P.
        • Tseng S.C.
        Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation.
        Invest Ophthalmol Vis Sci. 2001; 42: 1969-1974
        • Bauer D.
        • Wasmuth S.
        • Hermans P.
        • et al.
        On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation.
        Exp Eye Res. 2007; 85: 335-345
        • Kim J.S.
        • Kim J.C.
        • Na B.K.
        • Jeong J.M.
        • Song C.Y.
        Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn.
        Exp Eye Res. 2000; 70: 329-337
        • Buddi R.
        • Lin B.
        • Atilano S.R.
        • Zorapapel N.C.
        • Kenney M.C.
        • Brown D.J.
        Evidence of oxidative stress in human corneal diseases.
        J Histochem Cytochem. 2002; 50: 341-351
        • Atilano S.R.
        • Chwa M.
        • Kim D.W.
        • et al.
        Hydrogen peroxide causes mitochondrial DNA damage in corneal epithelial cells.
        Cornea. 2009; 28: 426-433
        • Choi S.I.
        • Kim T.I.
        • Kim K.S.
        • et al.
        Decreased catalase expression and increased susceptibility to oxidative stress in primary cultured corneal fibroblasts from patients with granular corneal dystrophy type II.
        Am J Pathol. 2009; 175: 248-261
        • Ou J.
        • Walczysko P.
        • Kucerova R.
        • et al.
        Chronic wound state exacerbated by oxidative stress in Pax6+/- aniridia-related keratopathy.
        J Pathol. 2008; 215: 421-430
        • Lockington D.
        • Macdonald E.C.
        • Stewart P.
        • Young D.
        • Caslake M.
        • Ramaesh K.
        Free radicals and the pH of topical glaucoma medications: a lifetime of ocular chemical injury?.
        Eye (Lond). 2012; 26: 734-741
        • Lockington D.
        • Macdonald E.C.
        • Young D.
        • Stewart P.
        • Caslake M.
        • Ramaesh K.
        Presence of free radicals in intracameral agents commonly used during cataract surgery.
        Br J Ophthalmol. 2010; 94: 1674-1677
        • Lockington D.
        • Macdonald E.
        • Gregory M.
        • Stewart P.
        • Caslake M.
        • Ramaesh K.
        Presence of free radicals in commonly used ophthalmic preparations.
        Br J Ophthalmol. 2010; 94: 525-526
        • Kobayashi A.
        • Shirao Y.
        • Yoshita T.
        • et al.
        Temporary amniotic membrane patching for acute chemical burns.
        Eye (Lond). 2003; 17: 149-158
        • Yoon K.C.
        • Im S.K.
        • Kim J.C.
        • Yoon K.W.
        • Choi S.K.
        Prognosis of paraquat-induced ocular surface injury: therapeutic effect of amniotic membrane transplantation.
        Cornea. 2009; 28: 520-523
        • Saccà S.C.
        • Izzotti A.
        Oxidative stress and glaucoma: injury in the anterior segment of the eye.
        Prog Brain Res. 2008; 173: 385-407
        • Mathew M.C.
        • Ervin A.M.
        • Tao J.
        • Davis R.M.
        Antioxidant vitamin supplementation for preventing and slowing the progression of age-related cataract.
        Cochrane Database Syst Rev. 2012; 6: CD004567
        • Evans J.R.
        Antioxidant vitamin and mineral supplements for age-related macular degeneration.
        Cochrane Database Syst Rev. 2002; (CD000254)
        • Hopkinson A.
        • McIntosh R.S.
        • Tighe P.J.
        • James D.K.
        • Dua H.S.
        Amniotic membrane for ocular surface reconstruction: donor variations and the effect of handling on TGF-beta content.
        Invest Ophthalmol Vis Sci. 2006; 47: 4316-4322