Abstract
Advertisements targeted at the elderly population suggest that antioxidant therapy will reduce free radicals and promote wound healing, yet few scientific studies substantiate these claims. To better understand the potential utility of supplemental antioxidant therapy for wound healing, we tested the hypothesis that age and tissue ischemia alter the balance of endogenous antioxidant enzymes. Using a bipedicled skin flap model, ischemic and non-ischemic wounds were created on young and aged rats. Wound closure and the balance of the critical antioxidants superoxide dismutase and glutathione in the wound bed were determined. Ischemia delayed wound closure significantly more in aged rats. Lower superoxide dismutase 2 and glutathione in non-ischemic wounds of aged rats indicate a basal deficit due to age alone. Ischemic wounds from aged rats had lower superoxide dismutase 2 protein and activity initially, coupled with decreased ratios of reduced/oxidized glutathione and lower glutathione peroxidase activity. De novo glutathione synthesis, to restore redox balance in aged ischemic wounds, was initiated as evidenced by increased glutamate cysteine ligase. Results demonstrate deficiencies in two antioxidant pathways in aged rats that become exaggerated in ischemic tissue, culminating in profoundly impaired wound healing and prolonged inflammation.
Similar content being viewed by others
References
Ashcroft GS, Horan MA, Ferguson MW (1997a) Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol 108(4):430–437
Ashcroft GS, Horan MA, Herrick SE, Tarnuzzer RW, Schultz GS, Ferguson MW (1997b) Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans. Cell Tissue Res 290(3):581–591
Ashcroft GS, Horan MA, Ferguson MW (1998) Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing. Lab Invest 78(1):47–58
Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271(5 Pt 1):C1424–C1437
Bonomo SR, Davidson JD, Tyrone JW, Lin X, Mustoe TA (2000) Enhancement of wound healing by hyperbaric oxygen and transforming growth factor beta3 in a new chronic wound model in aged rabbits. Arch Surg 135(10):1148–1153
Chen CN, Brown-Borg HM, Rakoczy SG, Ferrington DA, Thompson LV (2010) Aging impairs the expression of the catalytic subunit of glutamate cysteine ligase in soleus muscle under stress. J Gerontol A Biol Sci Med Sci 65(2):129–137
Choksi KB, Boylston WH, Rabek JP, Widger WR, Papaconstantinou J (2004) Oxidatively damaged proteins of heart mitochondrial electron transport complexes. Biochim Biophys Acta 1688(2):95–101
Copin JC, Gasche Y, Chan PH (2000) Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase. Free Radic Biol Med 28(10):1571–1576
Davis CA, Hearn AS, Fletcher B, Bickford J, Garcia JE, Leveque V, Melendez JA, Silverman DN, Zucali J, Agarwal A, Nick HS (2004) Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition. J Biol Chem 279(13):12769–12776
Demicheli V, Quijano C, Alvarez B, Radi R (2007) Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide. Free Radic Biol Med 42(9):1359–1368
Franklin CC, Backos DS, Mohar I, White CC, Forman HJ, Kavanagh TJ (2009) Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase. Mol Aspects Med 30(1–2):86–98
Gould LJ, Leong M, Sonstein J, Wilson S (2005) Optimization and validation of an ischemic wound model. Wound Repair Regen 13(6):576–582
Greenacre SA, Rocha FA, Rawlingson A, Meinerikandathevan S, Poston RN, Ruiz E, Halliwell B, Brain SD (2002) Protein nitration in cutaneous inflammation in the rat: essential role of inducible nitric oxide synthase and polymorphonuclear leukocytes. Br J Pharmacol 136(7):985–994
Gurjala AN, Liu WR, Mogford JE, Procaccini PS, Mustoe TA (2005) Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence. Wound Repair Regen 13(6):565–575
Kim A, Murphy MP, Oberley TD (2005) Mitochondrial redox state regulates transcription of the nuclear-encoded mitochondrial protein manganese superoxide dismutase: a proposed adaptive response to mitochondrial redox imbalance. Free Radic Biol Med 38(5):644–654
Krueger KE, Srivastava S (2006) Posttranslational protein modifications: current implications for cancer detection, prevention, and therapeutics. Mol Cell Proteomics 5(10):1799–1810
Liu R, Choi J (2000) Age-associated decline in gamma-glutamylcysteine synthetase gene expression in rats. Free Radic Biol Med 28(4):566–574
Liu H, Wang H, Shenvi S, Hagen TM, Liu RM (2004) Glutathione metabolism during aging and in Alzheimer disease. Ann N Y Acad Sci 1019:346–349
Lizarbe TR, Garcia-Rama C, Tarin C, Saura M, Calvo E, Lopez JA, Lopez-Otin C, Folgueras AR, Lamas S, Zaragoza C (2008) Nitric oxide elicits functional MMP-13 protein-tyrosine nitration during wound repair. FASEB J 22(9):3207–3215
MacMillan-Crow LA, Crow JP, Thompson JA (1998) Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues. Biochemistry 37(6):1613–1622
Mogford JE, Sisco M, Bonomo SR, Robinson AM, Mustoe TA (2004) Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing. J Surg Res 118(2):190–196
Morrison JP, Coleman MC, Aunan ES, Walsh SA, Spitz DR, Kregel KC (2005) Aging reduces responsiveness to BSO- and heat stress-induced perturbations of glutathione and antioxidant enzymes. Am J Physiol Regul Integr Comp Physiol 289(4):R1035–R1041
Mustoe TA, O’Shaughnessy K, Kloeters O (2006) Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 117(7 Suppl):35S–41S
Paglia DE, Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70(1):158–169
Radi R, Beckman JS, Bush KM, Freeman BA (1991) Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288(2):481–487
Salgo MG, Bermudez E, Squadrito GL, Pryor WA (1995a) Peroxynitrite causes DNA damage and oxidation of thiols in rat thymocytes [corrected]. Arch Biochem Biophys 322(2):500–505
Salgo MG, Squadrito GL, Pryor WA (1995b) Peroxynitrite causes apoptosis in rat thymocytes. Biochem Biophys Res Commun 215(3):1111–1118
Seelig GF, Simondsen RP, Meister A (1984) Reversible dissociation of gamma-glutamylcysteine synthetase into two subunits. J Biol Chem 259(15):9345–9347
Sen CK (2003) The general case for redox control of wound repair. Wound Repair Regen 11(6):431–438
Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H (1994) Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev 74(1–2):121–133
Soybir OC, Gurdal SO, Oran ES, Tulubas F, Yuksel M, Akyildiz AI, Bilir A, Soybir GR (2012) Delayed cutaneous wound healing in aged rats compared to younger ones. Int Wound J 9(5):478–487
Swift ME, Burns AL, Gray KL, DiPietro LA (2001) Age-related alterations in the inflammatory response to dermal injury. J Invest Dermatol 117(5):1027–1035
Szczesny B, Bhakat KK, Mitra S, Boldogh I (2004) Age-dependent modulation of DNA repair enzymes by covalent modification and subcellular distribution. Mech Ageing Dev 125(10–11):755–765
Tu Z, Anders MW (1998) Expression and characterization of human glutamate-cysteine ligase. Arch Biochem Biophys 354(2):247–254
Wu L, Brucker M, Gruskin E, Roth SI, Mustoe TA (1997) Differential effects of platelet-derived growth factor BB in accelerating wound healing in aged versus young animals: the impact of tissue hypoxia. Plast Reconstr Surg 99(3):815–822, discussion 823–814
Wu L, **a YP, Roth SI, Gruskin E, Mustoe TA (1999) Transforming growth factor-beta1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: importance of oxygen and age. Am J Pathol 154(1):301–309
Zelko IN, Mariani TJ, Folz RJ (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 33(3):337–349
Acknowledgments
This work was supported by a Veteran’s Affairs Merit Award (BX09-002) to L. Gould. The authors would like to thank the University of South Florida Pathology Core Facility for the preparation of histological specimens. The authors declare no conflicts of interest for this work.
Disclaimer
The contents of this manuscript do not represent the views of the Department of Veterans Affairs or the US Government.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Online Resource 1
Representative H&E staining of days 10, 14, and 21 non-ischemic (a–c young; d–f aged) wounds at 4× power and 40× insets (n = 3–4 sections/group/time point). TE thickened epidermis. Scale bars = 500 μm and 50 μM (insets; JPEG 212 kb)
About this article
Cite this article
Moor, A.N., Tummel, E., Prather, J.L. et al. Consequences of age on ischemic wound healing in rats: altered antioxidant activity and delayed wound closure. AGE 36, 733–748 (2014). https://doi.org/10.1007/s11357-014-9617-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11357-014-9617-4