Carnitine reduces the lipoperoxidative damage of the membrane and apoptosis after induction of cell stress in experimental glaucoma

N Calandrella, C De Seta, G Scarsella, G Risuleo, N Calandrella, C De Seta, G Scarsella, G Risuleo

Abstract

The pathological damage caused by glaucoma is associated to a high intraocular pressure. The ocular hypertone is most likely due to a defective efflux of aqueous humor from the anterior chamber of the eye. Ocular hypertension causes apoptotic death of retinal ganglion cells and overexpression of molecular markers typical of cell stress response and apoptosis. In this work, we report on the neuroprotective, antiapoptotic and antioxidant action of a natural substance, -carnitine. This compound is known for its ability to improve the mitochondrial performance. We analyze a number of cellular and molecular markers, typical of ocular hypertension and, in general, of the cell stress response. In particular, L-carnitine reduces the expression of glial fibrillary acidic protein, inducible nitric oxide synthase, ubiquitin and caspase 3 typical markers of cell stress. In addition, the morphological analysis of the optic nerve evidenced a reduction of the pathological excavation of the optic disk. This experimental hypertone protocol induces a severe lipoperoxidation, which is significantly reduced by L-carnitine. The overall interpretation is that mortality of the retinal cells is due to membrane damage.

Figures

Figure 1
Figure 1
Reduction of the expression of stress markers at retinal level induced by carnitine. (a) Immunolocalization of GFAP assessed by immunofluorescence in rat retina. 1: retina from untreated control eye. 2: Contralateral eye treated with methylcellulose in the presence of carnitine. 3: Eye treated only with methylcellulose. Magnification was × 500 in all cases. (b) Western blot on total protein from retina extracts to assess the expression of iNOS, ubiquitin and α-tubulin as standard reference. In this and subsequent figures, lanes 1, 2, and 3 report the results obtained in the same experimental conditions as in panel a; that is: control, MTC plus -carnitine and MTC alone
Figure 2
Figure 2
Carnitine reduces apoptotic cell death consequent to the ocular hypertone. (a) Evaluation of DNA damage by TUNEL assay. Magnification was × 500 in all cases. (b) Western blot on retina total protein extracts to assess the expression of caspase 3. Data shown in 1, 2 and 3 report in the same order the results obtained in the experimental conditions as in Figure 1
Figure 3
Figure 3
Morphological alterations of cup-to-eye. Control retinal samples (1); methylcellulose treated retinas in the presence (2); or absence of carnitine (3). Magnification was × 500 in all cases
Figure 4
Figure 4
Level of membrane lipoperoxidation in retina samples and role of -carnitine in the control of apoptosis. (a) Bars report the level of malondialdehyde (MDA) evaluated as indicated at the bottom of each bar. Mtc, methylcellulose; Carn, -carnitine. (b) The increase of the intraocular pressure (IOP) results in oxidative stress as shown by the overexpression of inducible nitric oxide synthase (iNOS). Mitochondrial lipid peroxidation causes the accumulation of intracellular MDA: a hallmark of lipoperoxidation. The activation of the ubiquitin (Ub)-mediated proteasome pathway is directly related to the execution of the apoptotic death as also shown by the stimulation of caspase 3 expression. Treatment with methylcellulose in the presence of carnitine reduces the level of all markers of apoptosis, and therefore -carnitine also improves the overall homeostatic response and limits apoptotic phenomena

References

    1. Cedrone C, Mancino R, Cerulli A, Cesareo M, Nucci C. Epidemiology of primary glaucoma: prevalence, incidence, and blinding effects. Prog Brain Res. 2008;173:3–14.
    1. Kwon YH, Fingert JH, Kuehn MH, Alward WL. Primary open-angle glaucoma. N Engl J Med. 2009;360:1113–1124.
    1. Friedman DS, Wilson MR, Liebmann JM, Fechtner RD, Weinreb RN. An evidence-based assessment of risk factors for the progression of ocular hypertension and glaucoma. Am J Ophthalmol Rev. 2004;138:19–31.
    1. Quigley HA. Number of people with glaucoma worldwide. Br J Opthalmol. 1996;80:389–393.
    1. Morgan JE. Optic nerve head structure in glaucoma: astrocytes as mediators of axonal damage. Eye. 2000;14:437–444.
    1. Guo L, Moss SE, Alexander RA, Ali RR, Fitzke FW, Cordeiro MF. Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix. Invest Ophthalmol Vis Sci. 2005;46:175–182.
    1. Ji J, Chang P, Pennesi ME, Yang Z, Zhang J, Li D, Wu SM, Gross RL. Effects of elevated intraocular pressure on mouse retinal ganglion cells. Vision Res. 2005;45:169–179.
    1. Calandrella N, Scarsella G, Pescosolido N, Risuleo G. Degenerative and apoptotic events at retinal and optic nerve level after experimental induction of ocular hypertension. Mol Cell Biochem. 2007;301:155–163.
    1. Garcia-Valenzuela E, Shareef S, Walsh J, Sharm SC. Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp Eye Res. 1995;61:33–44.
    1. Neufeld AH, Liu B. Glaucomatous optic neuropathy: when glia misbehave. Neuroscientist. 2003;9:485–495.
    1. Nakazawa T, Nakazawa C, Matsubara A, Noda K, Hisatomi T, She H, et al. Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J Neurosci. 2006;26:2633–2641.
    1. Hernandez MR. The optic nerve head in glaucoma: role of astrocytes in tissue remodelig. Prog Retin Eye Res. 2000;3:297–321.
    1. Varela HJ, Hernandez MR. Astrocyte responses in human optic nerve head with primary open angle glaucoma. J Glaucoma. 1997;6:303–313.
    1. Ricard CS, Kobayashi S, Pena JDO, Salvador-Silva M, Agapova O, Hernandez MR. Selective expression of neuronal cell adhesion molecule (NCAM)-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro. Mol Brain Res. 2000;81:62–79.
    1. Okada M, Matsumura M, Ogino N. Müller cells in detached human retina express glial fibrillary acidic protein and vimentin, Graefes Arch. Clin Exp Ophthalmol. 1990;228:467–474.
    1. Wang X, Tay SSW, Ng YK. An immunohistochemical study of neuronal and glial cell reaction in retina of rats with experimental glaucoma. Exp Brain Res. 2000;132:476–484.
    1. Tezel G, Hernandez MR, Wax BM. In vitro evaluation of reactive astrocyte migration, a component of tissue component remodeling in glaucomatous optic nerve head. Glia. 2001;3:178–189.
    1. Neufeld A, Hernandez MR, Gonzales M. Nitric oxide sinthase in the human glaucomatous optic nerve head. Arch Ophthalmol. 1997;115:497–503.
    1. Neufeld AH. Nitric oxide: a potential mediator of retinal ganglion cell damage in glaucoma. Surv Ophthalmol. 1999;43:129–135.
    1. Garthwaite G, Goodwin DA, Batchelor AM, Leeming K, Garthwaite J. Nitric oxide toxicity in CNS white matter: an in vitro study using rat optic nerve. Neuroscience. 2002;109:145–155.
    1. Dalkara T, Endres M, Moskowitz MA. Mechanisms of NO neurotoxicity. Prog Brain Res. 1998;118:231–239.
    1. Ullrich V, Bachschmid M. Superoxide as a messenger of endothelial function. Biochem Biophys Res Commun. 2000;11:1–8.
    1. Schneemann A, Leusink-Muis A, van den Berg TJ, Hoyng PF, Kamphuis W. Elevation of nitric oxide production in human trabecular meshwork by increased pressure. Graefes Arch Clin Exp Ophthalmol. 2003;24:321–326.
    1. Motallebipour M, Rada-Iglesias A, Jansson M, Wadelius C. The promoter of inducible nitric oxide synthase implicated in glaucoma based on genetic analysis and nuclear factor binding. Mol Vis. 2005;11:950–957.
    1. Fernandes R, Ramalho J, Pereira P. Oxidative stress upregulates the ubiquitin proteasome pathway in retinal endothelial cells. Mol Vis. 2006;12:1526–1535.
    1. Bresin A, Iacoangeli A, Risuleo G, Scarsella G. Ubiquitin depend proteolysis is activated in apoptotic fibroblasts in culture. Mol Cell Biochem. 2001;220:57–60.
    1. Lee JC, Peter ME. Regulation of apoptosis by ubiquitination. Immunol Rev. 2003;193:39–47.
    1. Risuleo G, Cristofanilli M, Scarsella G. Acute ischemia/hypoxia in rat hippocampal neurons activates nuclear ubiquitin and alters chromatin and DNA. Mol Cell Biochem. 2003;250:73–80.
    1. Ishii T, Shimpo Y, Matsuoka Y, Kinoshita K. Anti-apoptotic effect of acetyl--carnitine and -carnitine in primary cultured neurons. Jpn J Pharmacol. 2000;83:119–124.
    1. Cifone MG, Alesse E, Di Marzio L, Ruggeri B, Zazzeroni F, et al. Effect of -carnitine treatment in vivo on apoptosis and ceramide generation in peripheral blood lymphocytes from AIDS patients. Proc Assoc Physicians. 1997;109:146–153.
    1. Mutomba MC, Yuan M, Konyavko M, Adachi S, Yokoyama CB, Esser V, et al. Regulation of the activity of caspase by -carnitine and palmitoylcarnitine. FEBS Lett. 2000;478:19–25.
    1. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770–776.
    1. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med. 2000;6:513–519.
    1. Beal MF. Bioenergetic approaches for neuroprotection in Parkinson's disease. Ann Neurol. 2003;53:39–47.
    1. Binienda Z. Neuroprotective effects of - carnitine in induced mitochondrial dysfunction. Ann NY Acad Sci. 2003;993:289–295.
    1. Folts JD, Shung AL, Koke JR, Bittar N. Protection of the ischemic dog myocardium with carnitine. Am J Cardiol. 1978;41:1209–1214.
    1. Moretti S, Famularo G, Marcellini S, Boschini A, Santini G, Trinchieri V, et al. - carnitine reduces lymphocyte apoptosis and oxidant stress in HIV-1-infected subyects treated with Zidovudine and Dianosine. Antiox and Redox Sig. 2002;4:391–403.
    1. Pillich RT, Scarsella G, Risuleo G. Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl--carnitine to mouse fibroblasts in culture. Exp Cell Res. 2005;306:1–8.
    1. Alagoz G, Celiker U, Ilhan N, Yekeler H, Demir T, Celiker H. -carnitine in experimental retinal ischemia-reperfusion injury. Ophthalmol. 2002;216:144–150.
    1. Hewitson TD, Bisucci T, Darby IA. Histochemical localization of apoptosis with in situ labeling of fragmented DNA. Methods Mol Biol. 2006;326:227–234.

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit