The temporal and spatial profiles of cell loss following experimental spinal cord injury: effect of antioxidant therapy on cell death and functional recovery

BMC Neuroscience

Danxia Liu¹  Hao Qian²  Feng Bao²  Xiang Ling² 

[1] Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Rt. 0881, Galveston, TX 77555-0881, USA;Department of Neurology, University of Texas Medical Branch, 301 University Blvd., Rt. 0881, Galveston, TX 77555-0881, USA

关键词: Secondary spinal cord injury;    Nitro-L-arginine;    Mn (III) tetrakis (4-benzoic acid) porphyrin;    Behavioral test;    Apoptotic cell death;    Antioxidant therapy;   

Others  :  1131189
DOI  :  10.1186/1471-2202-14-146

 received in 2013-05-23, accepted in 2013-11-12,  发布年份 2013

【 摘 要 】

Background

Traumatic spinal cord injury (SCI)-induced overproduction of endogenous deleterious substances triggers secondary cell death to spread damage beyond the initial injury site. Substantial experimental evidence supports reactive species (RS) as important mediators of secondary cell death after SCI. This study established quantitative temporal and spatial profiles of cell loss, characterized apoptosis, and evaluated the effectiveness of a broad spectrum RS scavenger - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and a combination of MnTBAP plus nitro-L-arginine to prevent cell loss and neurological dysfunction following contusion SCI to the rat spinal cord.

Results

By counting the number of surviving cells in spinal cord sections removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI and at 0 – 4 mm from the epicenter, the temporal and spatial profiles of motoneuron and glia loss were established. Motoneurons continued to disappear over a week and the losses decreased with increasing distance from the epicenter. Significant glia loss peaked at 24 to 48 h post-SCI, but only at sections 0–1.5 mm from the epicenter. Apoptosis of neurons, motoneurons and astrocytes was characterized morphologically by double immuno-staining with cell-specific markers and apoptosis indicators and confirmed by transmission electron microscopy. DNA laddering, ELISA quantitation and caspase-3 activation in the spinal cord tissue indicated more intense DNA fragments and greater caspase-3 activation in the epicenter than at 1 and 2 cm away from the epicenter or the sham-operated sections. Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group. MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.

Conclusions

Our temporal and spatial profiles of cell loss provide data bases for determining the time and location for pharmacological intervention. Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction. MnTBAP significantly improved functional recovery, which strongly supports the important role of antioxidant therapy in treating SCI and the candidacy of MnTBAP for such treatment.

【 授权许可】

   
2013 Ling et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

【 参考文献 】

• [1]Young W: Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 1993, 11(Suppl 1):13-22.
• [2]Choo AM, Liu J, Dvorak M, Tetzlaffc W, Oxland TR: Secondary pathology following contusion, dislocation, and distraction spinal cord injuries. Exp Neurol 2008, 212:490-506.
• [3]Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS: Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med 1997, 3:73-76.
• [4]Liu XZ, Xu XM, Hu R, Du C, Zhang SX, McDonald JW, Dong HX, Wu YJ, Fan GS, Jacquin MF, et al.: Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 1997, 17:5395-5406.
• [5]Yong C, Arnold PM, Zoubine MN, Citron BA, Watanabe I, Berman NE, Festoff BW: Apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma 1998, 15:459-472.
• [6]Moon JY, Lee JY, Oh MS, Pak YK, Park K, Oh TH, Yune TY: Inhibition of inflammation and oxidative stress by angelica dahuricae radix extract decreases apoptotic cell death and improves functional recovery after spinal cord injury. J Neurosci Res 2012, 90:243-256.
• [7]Lewen A, Matz P, Chan PH: Free radical pathways in CNS injury. J Neurotrauma 2000, 17:871-890.
• [8]Chan PH: Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cerebr Blood F Met 2001, 21:2-14.
• [9]Genovese T, Cuzzocrea S: Role of free radicals and poly(ADP-ribose)polymerase-1 in the development of spinal cord injury: new potential therapeutic targets. Curr Med chem 2008, 15:477-487.
• [10]Genovese T, Mazzon E, Esposito E, Muia C, Di Paola R, Bramanti P, Cuzzocrea S: Beneficial effects of FeTSPP, a peroxynitrite decomposition catalyst, in a mouse model of spinal cord injury. Free Radic Biol Med 2007, 43:763-780.
• [11]Genovese T, Mazzon E, Esposito E, Di Paola R, Murthy K, Neville L, Bramanti P, Cuzzocrea S: Effects of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in a mouse model of spinal cord injury. Free radic res 2009, 43:631-645.
• [12]Yune TY, Lee JY, Jiang MH, Kim DW, Choi SY, Oh Tae H: Systematic administration of PEP-1-SOD1 fusion protein improves functional recovery by inhibition of neuronal cell death after spinal cord injury. Free Radic Biol Med 2008, 45:1190-1200.
• [13]Yune TY, Lee JY, Cui CM, Kim HC, Oh TH: Neuroprotective effect of Scutellaria baicalensis on spinal cord injury in rats. J Neurochem 2009, 110:1276-1287.
• [14]Liu D, Sybert TE, Qian H, Liu J: Superoxide production after spinal injury detected by microperfusion of cytochrome c. Free Radic Biol Med 1998, 25:298-304.
• [15]Liu D, Liu J, Wen J: Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton reaction. Free Radic Biol Med 1999, 27:478-482.
• [16]Liu D, Liu J, Sun D, Wen J: The time course of hydroxyl radical formation following spinal cord injury: the possible role of the iron-catalyzed Haber-Weiss reaction. J Neurotrauma 2004, 21:805-816.
• [17]Liu D, Liu J, Sun D, Alcock NW, Wen J: Spinal cord injury increases iron levels: catalytic production of hydroxyl radicals. Free Radic Biol Med 2003, 34:64-71.
• [18]Liu D, Ling X, Wen J, Liu J: The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein. J Neurochem 2000, 75:2144-2154.
• [19]Chatzipanteli K, Garcia R, Marcillo AE, Loor KE, Kraydieh S, Dietrich WD: Temporal and segmental distribution of constitutive and inducible nitric oxide synthases after traumatic spinal cord injury: effect of aminoguanidine treatment. J Neurotrauma 2002, 19:639-651.
• [20]Scott GS, Jakeman LB, Stokes BT, Szabo C: Peroxynitrite production and activation of poly (adenosine diphosphate-ribose) synthetase in spinal cord injury. Ann Neurol 1999, 45:120-124.
• [21]Leski ML, Bao F, Wu L, Qian H, Sun D, Liu D: Protein and DNA oxidation in spinal injury: neurofilaments–an oxidation target. Free Radi. Biol Med 2001, 30:613-624.
• [22]Aksenova M, Butterfield DA, Zhang SX, Underwood M, Geddes JW: Increased protein oxidation and decreased creatine kinase BB expression and activity after spinal cord contusion injury. J Neurotrauma 2002, 19:491-502.
• [23]Carrico KM, Vaishnav R, Hall ED: Temporal and spatial dynamics of peroxynitrite-induced oxidative damage after spinal cord contusion injury. J Neurotrauma 2009, 26:1369-1378.
• [24]Qian H, Liu D: The time course of malondialdehyde production following impact injury to rat spinal cord as measured by microdialysis and high pressure liquid chromatography. Neurochem Res 1997, 22:1231-1236.
• [25]Springer JE, Azbill RD, Mark RJ, Begley JG, Waeg G, Mattson MP: 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake. J Neurochem 1997, 68:2469-2476.
• [26]Christie SD, Comeau B, Myers T, Sadi D, Purdy M, Mendez I: Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone. Neurosurg Focus 2008, 25:E5.
• [27]Bao F, DeWitt DS, Prough DS, Liu D: Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn (III) tetrakis (4-benzoic acid) porphyrin. J Neurosci Res 2003, 71:220-227.
• [28]Liu D, Bao F, Prough DS, Dewitt DS: Peroxynitrite generated at the level produced by spinal cord injury induces peroxidation of membrane phospholipids in normal rat cord: reduction by a metalloporphyrin. J Neurotrauma 2005, 22:1123-1133.
• [29]Bao F, Liu D: Peroxynitrite generated in the rat spinal cord induces neuron death and neurological deficits. Neuroscience 2002, 115:839-849.
• [30]Bao F, Liu D: Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3. Neuroscience 2003, 116:59-70.
• [31]Bao F, Liu D: Hydroxyl radicals generated in the rat spinal cord at the level produced impact injury induce cell death by necrosis and apoptosis: protection by a metalloporphyrin. Neuroscience 2004, 126:285-295.
• [32]Day BJ: Catalytic antioxidants: a radical approach to new therapeutics. Drug Discov Toda 2004, 9:557-566.
• [33]Batinic-Haberle I, Reboucas JS, Spasojevic I: Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential. Antioxid Redox Signal 2010, 13:877-918.
• [34]Day BJ, Fridovich I, Crapo JD: Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury. Arch Biochem Biophys 1997, 347:256-262.
• [35]Crow JP: Peroxynitrite scavenging by metalloporphyrins and thiolates. Free Radic Boil Med 2000, 28:1487-1494.
• [36]Day BJ, Batinic-Haberle I, Crapo JD: Metalloporphyrins are potent inhibitors of lipid peroxidation. Free Radic Biol Med 1999, 26:730-736.
• [37]Liang LP, Ho YS, Patel M: Mitochondrial superoxide production in kainate-induced hippocampal damage. Neuroscience 2000, 101:563-570.
• [38]Hachmeister JE, Valluru L, Bao F, Liu D: Mn (III) tetrakis (4-benzoic acid) porphyrin administered into the intrathecal space reduces oxidative damage and neuron death after spinal cord injury: a comparison with methylprednisolone. J Neurotrauma 2006, 23:1766-1778.
• [39]Valluru L, Diao Y, Hachmeister JE, Liu D: Mn (III) tetrakis (4-benzoic acid) porphyrin protects against neuronal and glial oxidative stress and death after spinal cord injury. CNS Neurol Disord Drug Targets 2012, 11:774-790.
• [40]Liu D, Shan Y, Valluru L, Bao F: Mn (III) tetrakis (4-benzoic acid) porphyrin scavenges reactive species, reduces oxidative stress, and improves functional recovery after experimental spinal cord injury in rats: comparison with methylprednisolone. BMC Neurosci 2013, 14:23. doi: 10.1186/1471-2202-14-23. URL: http://www.biomedcentral.com/1471-2202/14/23 webcite
• [41]Melov S, Schneider JA, Day BJ, Hinerfeld D, Coskun P, Mirra SS, Crapo JD, Wallace DC: A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase. Na Genet 1998, 18:159-163.
• [42]Wu L, Shan Y, Liu D: Stability, disposition, and penetration of catalytic antioxidants Mn-porphyrin and Mn-salen and of methylprednisolone in spinal cord injury. CNS Agent Med Chem 2012, 12:122-130.
• [43]Ling X, Liu D: Temporal and spatial profiles of cell loss after spinal cord injury: reduction by a metalloporphyrin. J Neurosci Res 2007, 85:2175-2185.
• [44]Szabo C, Day BJ, Salzman AL: Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondiral respiration in immunostimulated macrophages using a manganese mesopophyrin superoxide dismutase mimetic and peroxynitrite scavenger. FEBS Lett 1996, 381:82-86.
• [45]Allen AR: Surgery of experimental spinal cord injury equivalent to crush injury of fracture dislocation of spinal column. JAMA 1911, 57:878-880.
• [46]Jacob JM: Lumbar motor neuron size and number is affected by age in male F344 rats. Mech Ageing Dev 1998, 106:205-216.
• [47]Taglialatel G, Gegg M, Perez-Polo JR, Williams LR, Rose GM: Evidence for DNA fragmentation in the CNS of aged Fisher-344 rats. Neuro Report 1996, 7:977-980.
• [48]Beland FA, Dooley KL, Casciano DA: Rapid isolation of carcinogen-bound DNA and RNA by hydroxyapatite chromatography. J Chromatogr 1979, 174:177-186.
• [49]Cregan SP, Maclaurin JG, Craig CG, Robertson GS, Nicholson DW, Park DS, Slack RS: Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons. J Neurosci 1999, 19:7860-7869.
• [50]Fukuzuka K, Rosenberg JJ, Gaines GC, Edwards CK 3rd, Clare-Salzler M, MacKay SL, Moldawer LL, Copeland EM 3rd, Mozingo DW: Caspase-3-dependent organ apoptosis early after burn injury. Ann Surg 1999, 229:851-858. discussion 858–859
• [51]Lord-Fontaine S, Yang F, Diep Q, Dergham P, Munzer S, Tremblay P, McKerracher LJ: Local inhibition of Rho signaling by cell-permeable recombinant protein BA-210 prevents secondary damage and promotes functional recovery following acute spinal cord injury. J Neurotrauma 2008, 25:1309-1322.
• [52]Santiago JM, Rosas O, Torrado AI, González MM, Kalyan-Masih PO, Miranda JD: Molecular, anatomical, physiological, and behavioral studies of rats treated with buprenorphine after spinal cord injury. J Neurotrauma 2009, 26:1783-93.
• [53]Yu CG, Joshi A, Geddes JW: Intraspinal MDL28170 microinjection improves functional and pathological outcome following spinal cord injury. J Neurotrauma 2008, 25:833-40.
• [54]Springer JE, Rao RR, Lim HR, Cho SI, Moon GJ, Lee HY, Park EJ, Noh JS, Gwag BJ: The functional and neuroprotective actions of Neu 2000, a dual-acting pharmacological agent, in the treatment of acute spinal cord injury. J Neurotrauma 2010, 27:139-49.
• [55]Durham-Lee JC, Mokkapati VU, Johnson KM, Nesic O: Amiloride improves locomotor recovery after spinal cord injury. J Neurotrauma 2011, 28:1319-26.
• [56]Basso DM, Beattie MS, Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995, 12:1-21.
• [57]Basso DM, Beattie MS, Bresnahan JC: Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 1996, 139:244-256.
• [58]Rivlin AS, Tator CH: Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg Neurol 1978, 10:38-43.
• [59]Rivlin AS, Tator CH: Effect of vasodilators and myelotomy on recovery after acute spinal cord injury in rats. J Neurosurg 1979, 50:349-352.
• [60]Von Euler M, Akesson E, Samuelsson EB, Seiger A, Sundstrom E: Motor performance score: a new algorithm for accurate behavioral testing of spinal cord injury in rats. Exp Neurol 1996, 137:242-254.
• [61]Grossman SD, Rosenberg LJ, Wrathall JR: Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp Neurol 2001, 168:273-282.
• [62]Bresnahan JC, Beattie MS, Stokes BT, Conway KM: Three-dimensional computer-assisted analysis of graded contusion lesions in the spinal cord of the rat. J Neurotrauma 1991, 8:91-101.
• [63]Hyun JK, Lee YI, Son YJ, Park JS: Serial changes in bladder, locomotion, and levels of neurotrophic factors in rats with spinal cord contusion. J Neurotrauma 2009, 26:1773-1782.
• [64]Wood KA, Youle RJ: Apoptosis and free radicals. Ann NY Acad Sci 1994, 738:400-407.
• [65]Beer R, Franz G, Srinivasan A, Hayes RL, Pike BR, Newcomb JK, Zhao X, Schmutzhard E, Poewe W, Kampfl A: Temporal profile and cell subtype distribution of activated caspase-3 following experimental traumatic brain injury. J Neurochem 2000, 75:1264-1273.
• [66]Sastry PS, Rao KS: Apoptosis and the nervous system. J Neurochem 2000, 74:1-20.
• [67]Eldadah BA, Faden AI: Caspase pathways, neuronal apoptosis, and CNS injury. J Neurotrauma 2000, 17:811-829.
• [68]Springer JE, Azbill RD, Knapp PE: Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat Med 1999, 5:943-946.
• [69]Xu W, Chi L, Xu R, Ke Y, Luo C, Cai J, Qiu M, Gozal D, Liu R: Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury. Spinal cord 2005, 43:204-213.
• [70]Impellizzeri D, Esposito E, Mazzon E, Paterniti I, Di Paola R, Bramanti P, Morittu VM, Procopio A, Perri E, Britti D, Cuzzocrea S: The effects of a polyphenol present in olive oil, oleuropein aglycone, in an experimental model of spinal cord injury in mice. Biochem Pharmacol 2012, 83:1413-1426.
• [71]Ning B, Zhang A, Song H, Gong W, Ding Y, Guo S, Zhao Y, Jiang J, Jia T: Recombinant human erythropoietin prevents motor neuron apoptosis in a rat model of cervical sub-acute spinal cord compression. Neurosci Lett 2011, 490:57-62.
• [72]Genovese T, Mazzon E, Esposito E, Muia C, Di Paola R, Di Bella P, Bramanti P, Cuzzocrea S: Role of endogenous glutathione in the secondary damage in experimental spinal cord injury in mice. Neurosci Lett 2007, 423:41-46.
• [73]Paterniti I, Mazzon E, Emanuela E, Paola RD, Galuppo M, Bramanti P, Cuzzocrea S: Modulation of inflammatory response after spinal cord trauma with deferoxamine, an iron chelator. Free Radic Res 2010, 44:694-709.
• [74]Impellizzeri D, Mazzon E, Esposito E, Paterniti I, Bramanti P, Cuzzocrea S: Effect of Apocynin, an inhibitor of NADPH oxidase, in the inflammatory process induced by an experimental model of spinal cord injury. Free Radic Res 2011, 45:221-236.
• [75]Di Paola R, Impellizzeri D, Salinaro AT, Mazzon E, Bellia F, Cavallaro M, Cornelius C, Vecchio G, Calabrese V, Rizzarelli E, Cuzzocrea S: Administration of carnosine in the treatment of acute spinal cord injury. Biochem Pharmacol 2011, 82:1478-1489.
• [76]Lee BI, Chan PH, Kim GW: Metalloporphyrin-based superoxide dismutase mimic attenuates the nuclear translocation of apoptosis-inducing factor and the subsequent DNA fragmentation after permanent focal cerebral ischemia in mice. Stroke 2005, 36:2712-2717.