- Research article
- Open Access
Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector
© Peluffo et al; licensee BioMed Central Ltd. 2006
Received: 11 November 2005
Accepted: 25 April 2006
Published: 25 April 2006
Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated.
Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced.
When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.
The pathobiology of acute damage to the CNS includes production of the superoxide anion (O2-.) and other reactive oxygen species that rapidly induce oxidative injury by lipid peroxidation, DNA damage and protein nitration  Many studies in the last decade have focused on the study of antioxidant proteins dealing with oxidative stress in physiological conditions and after injury.
Superoxide dismutases are among the most important cellular mechanisms that cope with oxidative stress. In normal conditions, cytosolic copper zinc superoxide dismutase (Cu/Zn SOD) and mitochondrial manganese superoxide dismutase (Mn SOD) are responsible for maintaining low levels of intracellular O2-. by catalyzing its dismutation to oxygen and H2O2 . In neuronal cells, endogenous Cu/Zn SOD is normally expressed but is rapidly downregulated after several types of acute brain insults [3–6] rendering the brain more susceptible to oxidative stress. In agreement with its antioxidant role, over-expression of Cu/Zn SOD in adult transgenic rats show pronounced neuroprotection in most acute CNS injury models [7–9] and targeted deletions of the Cu/Zn SOD gene or extracellular SOD genes worsens the outcome after focal ischemia in the adult brain [10, 11].
After hypoxic/ischemic injury to the immature CNS, contradictory results have been reported in regards to the role of Cu/Zn SOD. Brain damage as a result of perinatal hypoxic-ischemic insult is a serious clinical problem with severe neurological consequences, where oxidative stress is known to play a fundamental role [12, 13]. In previous studies using N-methyl-D-aspartate (NMDA)-mediated excitotoxicity, a model for hypoxic-ischemic injury to the postnatal brain [14, 15], we have shown an upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)  and increased levels of the oxidative marker nitrotyrosine . However, while a slightly worsened neuropathological outcome was observed in postnatal transgenic mice over-expressing Cu/Zn SOD [18, 19], several antioxidant molecules including SOD mimetics like O2-. dismuting metalloporphyrins have been shown to be neuroprotective . In this sense, several differences between immature and adult animals in terms of oxidative stress and antioxidant defenses have been described including: upregulation of gluthathione peroxidase in the damaged adult brain but not in the damaged immature brain after trauma , the rapid free iron accumulation within 4 hours after transient cerebral ischemia stimulating Fenton reactions in the immature brain [22, 23], the lesser concentration of metallothioneins, potent metal-binding antioxidant enzymes in the immature brain [24, 25]. Finally, the postnatal brain is more sensitive than the adult brain to the neurotoxic actions of NMDA  which will lead to increased O2-. generation [27, 28]. In view of these contradictory findings of Cu/Zn SOD expression, we induced a transient post-injury overexpression of Cu/Zn SOD after excitotoxic damage to the immature rat brain using a novel non-viral gene therapy approach.
The design of non-viral vectors showing transgene expression in the brain has recently gained interest [29–32] due to the limitations imposed by viral vectors [33, 34]. Non-viral modular approaches for gene therapy vectors based on the combination of several functional domains in a single polypeptide chain are of particular interest because of recombinant DNA methodologies that allow tailored designed vectors. The production of such protein vectors also permits a convenient scaling up and the vehicles exhibit high stability suitable for therapeutic uses. We have previously reported the construction of a recombinant modular protein vector that combines different functional domains displayed by E. coli β-galactosidase then engineered to conveniently accommodate a poly-lysine tail with DNA condensing/attaching properties and an integrin targeting RGD motif with cell attachment and internalization properties [35, 36]. The resulting vector was capable of transferring a transgene to the intact and lesioned brain without any detectable acute inflammatory reaction or immune activation . We have further improved the transfection efficiency of this modular vector by introducing the nuclear localization motif of the SV40 virus, generating the vector NLSCt .
The aim of this study was to assess the potential efficacy of the recombinant modular protein vector NLSCt complexed to the Cu/Zn SOD gene to improve neurological outcome after an acute excitotoxic injury to the immature brain.
Lesion volume and neuronal cell death
The injection of NMDA into the sensorimotor cortex of the postnatal brain induces a well-characterized excitotoxic lesion  which includes the sensorimotor cortex, the dorso-medial striatum, and the rostral hippocampus.
To determine if the reduction in the neurodegenerative area observed after the treatment with the NLSCt vector carrying the control GFP transgene was due to the DNA molecule or intrinsic to the NLSCt vector, we injected lesioned rats with the naked NLSCt vector. Interestingly, the NLSCt vector alone induced a significant 31.2% reduction of lesion volume (data not shown), reproducing the results obtained after the treatment with the NLSCt vector carrying the GFP transgene.
This study shows for the first time that consistent functional and neuropathological recovery from acute immature brain damage can be achieved by post-lesion overexpression of the Cu/Zn SOD antioxidant enzyme delivered through a modular multifunctional protein vector. This treatment protected against excitotoxic damage, thought to be an important mechanism underlying neuronal death after hypoxic/ischemic injury to the neonate [13, 40], but also in acute adult brain neurodegenerative conditions such as stroke  and traumatic brain injury , as well as in chronic ones as Alzheimer's , Parkinson's  and Huntington's  disease.
Cu/Zn SOD as a therapeutic transgene
In this study, the Cu/Zn SOD transgene was expressed under the control of the cytomegalovirus immediate early promoter and as such, expressed in any cell type. We have previously shown that after postnatal excitotoxic injury, neurons, astrocytes and microglial cells are the main cell types showing transgene expression mediated by NLSCt-type vectors . Under physiological conditions, Cu/Zn SOD is mainly expressed in neurons [6, 46], however, in our experimental conditions several glial cell types are also transfected  and could indirectly mediate the neuroprotective effect. Noteworthy, O2-. produced after damage in both neurons and glial cells can be dismutated to H2O2 by Cu/Zn SOD activity, hindering the formation of the potent oxidant and protein nitrating agent peroxynitrite [47, 48]. The reduction in the levels of nitrotyrosine reported here support this mechanism of Cu/Zn SOD neuroprotection. Interestingly, there is a great deal of evidence that suggests that O2-./peroxinitrite species toxicity is higher than that of H2O2. A study of Cu/Zn SOD overexpressing astrocytes exposed to O2-. found that they had higher survival rates than control astrocytes even when glutathione peroxidase and catalase activities were blocked and GSH levels depleted . Cu/Zn SOD overexpressing astrocytes also survived better that control astrocytes after oxygen glucose deprivation, in the absence of glutathione peroxidase upregulation and with a lower catalase upregulation in comparison to control astrocytes . These Cu/Zn SOD overexpressing astrocytes, unlike controls, also maintained elevated GSH concentration. Peroxinitrite but not H2O2 has also been shown to trigger an in vitro reactive phenotype of astrocytes that is toxic for co-cultured motor neurons . These data suggest that overproduction of H2O2 is not a major factor in astrocytic injury. On the other hand, specific scavenging of O2-. can also increase neuronal survival under some pathologically relevant conditions. For example, motor neurons can be rescued from trophic factor withdrawal by liposome-mediated Cu/Zn SOD protein delivery  and synthetic SOD mimetics [53, 54]. Neuronal cultures can also be protected from excitotoxicity by SOD mimetics , adenovirally mediated overexpression of Cu/Zn SOD , or transgenically overexpressed Cu/Zn SOD [57, 58]. However, in some particular cases, Cu/Zn SOD overexpression can reduce neuronal survival during direct extracellular exposure to superoxide generators by a mechanism involving excess H2O2 accumulation , while protecting from direct H2O2 treatment . Finally, some research suggests that O2-. is more toxic than H2O2 by reacting with NO to form peroxinitrite [47, 48] as been shown by inhibition of NO production in neuronal cultures submitted to an excitotoxic damage. This treatment is sufficient for inducing neuroprotection [60–62], while O2-. and H2O2 are still being formed but will not be so toxic. Thus, the increase in Cu/Zn SOD expression in neurons and astrocytes most likely contributes to the neuroprotection observed in vivo.
Regarding lesion volume, we show a significant decrease in all NLSCt treated animals when compared to NMDA+saline injected animals, but no difference between these NLSCt treated groups. However, only the Cu/Zn SOD treated animals showed a significant reduction in lesion volume compared to NMDA alone lesioned animals, which is in fact the real control of the therapeutic gene therapy approach. It is important to consider the functional recovery induced by Cu/Zn SOD therapy in both of the neurological evaluation tests that could reflect increased preservation of functional synaptic contacts and white matter tracts. The reduction in the developmental increase in daily body weight observed in lesioned animals and GFP treated animals was not observed in Cu/Zn SOD treated animals, which also supports the general improvement of these animals.
In accordance with our findings, SOD mimetics (such as O2-. dismuting metalloporphyrins) were shown to be neuroprotective after ischemia in the immature brain . However, our results contrast with the previously reported exacerbation of hypoxic/ischemic injury occurring in immature transgenic mice over expressing Cu/Zn SOD . Though the reason for this difference is not clear, several reasons besides species specificity and lesion model could contribute to its explanation. In our experimental conditions, the NLSCt vector induced a transient and lower level of Cu/Zn SOD transgene expression compared to the higher and permanent expression found in transgenic mice. It has previously been shown that very high levels of Cu/Zn SOD, as those observed in transgenic animals, can produce alterations such as, an increase in basal lipid peroxidation , mitochondrial vaquolation [64, 65], abnormalities in neuromuscular junctions , or deficits in long-term potentiation (LTP) and spatial memory . Furthermore, after life-long overexpression of Cu/Zn SOD, compensatory changes in the basal levels or induction of other antioxidant enzymes like Mn SOD , heme oxygenase , or glutathione peroxidase  have been documented that provide an altered redox balance in transgenic animals.
In regards to expression levels, it has been reported that polyethylene glycol-conjugated Cu/Zn SOD treatment after focal ischemia showed a U-shaped dose-response curve, implying that the effective neuroprotective dose of this enzyme may be in fact concentration restricted . Therefore, several parameters like protein levels, time-course and cell population of Cu/Zn SOD expression could affect the overall outcome after the lesion and underlie the differences between gene therapy and transgenic mice approaches.
NLSCt vector for neuroprotective gene therapy
Studies showing phenotypic or functional effects derived from transgene expression in the CNS are generally absent . Regarding non-viral vectors, only one flexible liposome/antibody-conjugated non-viral vector has been reported to induce functional recovery, reversing motor abnormalities after a 6-hydroxydopamine striatal lesion .
Several multifunctional protein vectors have been developed by combining functional modules from different origins, driving the four main steps for successful DNA transfer to the cell nucleus: DNA ligation-condensation, cell attachment-internalization, endosome disruption-escaping, and nuclear import. Although many of these prototypes can transfect cells in culture, their efficiency in vivo is very limited (reviewed in ). We have previously shown that an earlier version of the NLSCt modular vector had a restricted capacity for transgene delivery to the intact brain. However, it was very efficient in conducting widespread transgene expression after an excitotoxic lesion , probably due to the disruption of the extracellular matrix and the small size of the vector/DNA complexes (20–40 nm diameter) . In this study, we show that NLSCt had a very high transfection efficiency, only 24 ng of NLSCt-coated Cu/Zn SOD plasmid was able to reduce oxidative stress and rescue neurons from cell death in different areas of the lesion border, that considerably reduced the total lesion volume and increased functional outcome. This transfection efficiency could be due to integrin mediated endocytosis and enhanced transit towards the nuclear compartment mediated by the SV40 viral nuclear localization sequence of NLSCt . It could also be due to massive neuronal endocytosis that reaches the nuclear compartment, a process previously described in neuronal cells within a few hours after injection of toxic and sub-toxic doses of NMDA or kainate [74, 75].
Transient Cu/Zn SOD expression induced by NLSCt-mediated gene therapy seems to be an important parameter in conferring neuroprotection. In the same experimental model as the one used here, and also using the transgene regulated by the CMV promoter, the expression of the GFP transgene was transient, almost disappearing at 7 days postinjury . Whereas a prolonged stable delivery of a transgene would be desirable for long-term improvement of pathologies such as inherited monogenic defects, transient transgene delivery may be more beneficial than a constitutive expression for therapeutic intervention after acute CNS damage.
Interestingly, both the NLSCt vector carrying a control transgene and the nude vector showed a significant grade of neuroprotection, indicating that the effect was intrinsic to the NLSCt vector. One of the bioactive motifs of NLSCt is the foot-and-mouth disease virus integrin-interacting RGD peptide, which interacts preferentially with different αV integrins . This may suggest that our results with the vector show a neuroprotective effect mediated by the interaction between the RGD motif of NLSCt and specific integrins through an unknown mechanism. This is supported by a recent study showing that blocking αD/β2 integrins is strongly neuroprotective after spinal cord injury . In addition, although the NLSCt vector inhibits the interaction of RGD dependent integrins with their natural extracellular matrix ligands, it could be directly activating integrin outside-in signaling events .
We show that transient overexpression of Cu/Zn SOD after an excitotoxic injury to the immature rat brain using a modular protein for non-viral gene delivery can be neuroprotective and improve functional outcome; signalling this vector as a promising tool for in vivo gene therapy strategies for acute CNS lesions.
Protein, DNA and protein-DNA complexes
Protein NLSCt is an engineered form of Escherichia coli beta-galactosidase that displays an integrin-targeted RGD motif. This segment, inserted between residues 249 and 250 of the bacterial enzyme, reproduces the cell-attachment region of the VP1 capsid protein of foot-and-mouth disease virus  that binds host cells preferentially by integrin αVβ3 but also by integrins αVβ6, αVβ5, and αVβ8 . The additional presence of a deca-lysine tail joined to the amino terminus of the construct and a still unidentified enzyme segment with nuclear targeting properties and the SV40 NLS at carboxi terminus of the recombinant protein  enables NLSCt to promote efficient DNA delivery. The NLSCt protein was produced in bacteria and purified from crude cell extracts as described previously . The human Cu/Zn SOD gene cloned into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) under the control of a cytomegalovirus immediate early promoter was used (generously provided by AG Estévez and JS Beckman). A red-shift variant of jellyfish Aequorea Victoria green fluorescent protein (GFP) gene encoded into plasmid pEGFP-C1 (Clontech, Palo Alto, CA, USA) under the control of the same cytomegalovirus promoter was used as a control. Protein and DNA complexes were formed by incubation in 0.9 % NaCl at room temperature for 1 hour at ratios of 0.03 μg DNA per μg of NLSCt protein. Details of complex formation are provided elsewhere .
Excitotoxic injury and treatment paradigm
Nine-day old Long-Evans black-hooded rat pups (15–20 gr., both sexes; Janvier, France) were used. All intracerebral injections were made into the right sensorimotor cortex at the level of the coronal suture (2 mm lateral of bregma and 0.5 mm depth) using a stereotaxic frame adapted for new-borns (Kopf Instruments) under isoflurane (Baxter International Inc.) anesthesia. Excitotoxic lesions were performed as previously described , by injecting 18.5 nmol of N-methyl-D-aspartate (NMDA) (Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.15 μl of saline solution (0.9 % NaCl) at a rate of 0.05 μl/min using an automatic injector. One microliter of either the NLSCt vector (0.8 μg/μl) carrying the control EGFP plasmid or the Cu/Zn SOD plasmid, the NLSCt vector alone (0.8 μg/μl), or the vehicle (NaCl 0.9 %) was injected 2 hours after the excitotoxic lesion at the same coordinates at 0.2 μl/min. After suture, pups were placed on a thermal pad for 2 hours at 36°C to maintain normothermia. Experimental animal work was conducted according to Spanish regulations in agreement with European Union directives. Experimental procedures were approved by the ethical commission of the Autonomous University of Barcelona. All efforts were made to minimize animal suffering.
Behavioural and neurological testing
Quantitative methods for the evaluation of adult motor performance  have been adapted here for postnatal pups (P9–P12). These methods of scoring gave consistent values for different observers. From day 1 until day 3 post-lesion, all rats were weighed and tested once a day in each of the following tasks: general motor activity, net turns and inclinated grid climbing. For the evaluation of general motor activity and net turns, rats were placed in an open field (70 cm × 70 cm) immediately after the separation from the mother and their spontaneous activity was recorded as number of new squares (10 × 10 cm) visited. Simultaneously, the total number of spontaneous complete turns (ipsilateral = positive and contralateral = negative) were recorded. As previously shown elsewhere, un-lesioned animals as well as saline injected animals did not show turning bias towards either side. In this test, lesioned rats show spontaneous turning bias towards the ipsilateral side while the lesion over-stimulates the ipsilateral parenchyma. After one day it disappears due to the complete destruction of neurons and then turning can only be observed by injection of amphetamine. The inclinated grid climbing test was performed by allowing the rats to climb an inclined grid (metal bars of 3.5 mm in diameter and separated by 7.5 mm) at an angle of 45°. Climbing the grid is a spontaneous response. However, in the few cases when a rat stayed still on the grid, it was removed and placed again on the grid. The total time climbing on the grid until five consecutive falls occurred was recorded each day for every rat. The task was interrupted after 5 falls or after 360 seconds on the grid. Unlesioned animals and saline injected animals consistently climbed for longer time periods than lesioned animals.
Histology and lesion volume measurement
Three days post-lesion, rats were anaesthetized and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed in the same fixative for 2 hours and sunk in a 30% sucrose solution before being frozen with dry CO2. Coronal sections of the entire brain (30-μm thick) were obtained using a Leitz cryostat. Parallel sections of the entire brain every 240 μm were collected directly on a slide, stained for Nissl and used for the measurement of the total lesioned area and total hemisphere area after high resolution digitizing. Using the analySIS® software, the pale Nissl stained lesioned area was quantified by parallel observation of the slides on a microscope. To avoid misinterpretations due to possible tissue edema or postmortem alterations during cutting or mounting, data are presented as percentage of the ipsilateral hemisphere.
Immunohistochemistry for nitrotyrosine and densitometrical analysis
Nitrotyrosine labeling, a marker of peroxynitrite and other reactive oxygen species formation, was used to evaluate the level of oxidative stress. Sections were processed for endogenous peroxidase inactivation and blocked for 1 hour in Tris-buffered saline (TBS, pH 7.4), 10 % fetal calf serum plus 1% Triton X-100. Sections were incubated overnight at 4°C in the same blocking solution with a primary antibody against nitrotyrosine (1:60)(06–284, Upstate Biotechnology, Lake Placid, NY, USA). After several washes they were incubated for 1 hour with biotinylated anti-rabbit (1:200, Amersham RPN-1004). Specific labeling was evidenced by incubation with avidin-peroxidase (1:400 Dakopatts P0364) for 1 hour and subsequent 3,3'-diaminobenzidine (DAB)-hydrogen peroxide developing procedure. For densitometrical measurements, digitalized images from ipsilateral lesion border (penumbra) and contralateral hippocampal CA1 regions were analyzed with the analySIS® software for total grey intensity. Data was expressed as the ratio between immunolabeling intensity in the injured and contralateral hemispheres.
Fluoro-Jade B staining
Neuronal degeneration was detected as previously described . Briefly, free-floating sections were mounted and air dried overnight. After dehydration in ethanol (30%, 50%, 70%, 96% and 100%) and rehydration (etanol 96% and 70%), sections were rinsed with distilled water and oxidized with MnO4K (0.06% in water, 15 min.). Then sections were rinsed with distilled water, incubated with Fluoro Jade B (Histo-Chem, Inc. Jefferson, USA) (0.0004% in water plus 1% acetic acid glacial, 20 min.), washed with distilled water, air dried and mounted.
All results are expressed as mean ± standard error mean (SEM). Six rat litters containing at least 3 different experimental groups were used. A total of 8 saline injected animals and 51 NMDA injected animals were used (NMDA only, n = 4; NMDA+saline, n = 14; NMDA+NLSCt/pSOD, n = 13; NMDA+NLSCt/pEGFP, n = 16; and NMDA+NLSCt, n = 4). ANOVA followed by Fisher's PLSD post-hoc test was used to determine significant differences (p < 0.05) in lesion volume, cell counts, nitrotyrosine densitometry and number of squares visited measurements. Repeated measures ANOVA followed by Fisher's PLSD post-hoc test were used to evaluate significant differences (p < 0.05) between groups in weight increase and in inclinated grid walking. Analysis of significant differences between groups in the measurements of the number of net turns was performed by ANOVA followed by Fisher's PLSD post-hoc test after ranking of the data.
We thank Miguel Angel Martil and Dolors Mulero for their excellent technical help, J.S. Beckman and A.G. Estevez for providing the SOD expression plasmid, R.M. Escorihuela for helpful discussions regarding behavioural tests, Maryam Faiz for help with the manuscript editing, and J. Giraldo for helpful discussions regarding statistical analysis. This work was supported by BFI2002-02079. HP holds a FI fellowship from the Generalitat de Catalunya and PG from the Ministry of Science.
- Halliwell B: Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging. 2001, 18 (9): 685-716. 10.2165/00002512-200118090-00004.View ArticlePubMedGoogle Scholar
- McCord JM, Fridovich I: Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969, 244 (22): 6049-6055.PubMedGoogle Scholar
- DeKosky ST, Abrahamson EE, Taffe KM, Dixon CE, Kochanek PM, Ikonomovic MD: Effects of post-injury hypothermia and nerve growth factor infusion on antioxidant enzyme activity in the rat: implications for clinical therapies. J Neurochem. 2004, 90 (4): 998-1004. 10.1111/j.1471-4159.2004.02575.x.View ArticlePubMedGoogle Scholar
- Liu XH, Kato H, Nakata N, Kogure K, Kato K: An immunohistochemical study of copper/zinc superoxide dismutase and manganese superoxide dismutase in rat hippocampus after transient cerebral ischemia. Brain Res. 1993, 625 (1): 29-37. 10.1016/0006-8993(93)90134-9.View ArticlePubMedGoogle Scholar
- Kim H, Bing G, Jhoo W, Ko KH, Kim WK, Suh JH, Kim SJ, Kato K, Hong JS: Changes of hippocampal Cu/Zn-superoxide dismutase after kainate treatment in the rat. Brain Res. 2000, 853 (2): 215-226. 10.1016/S0006-8993(99)02254-4.View ArticlePubMedGoogle Scholar
- Peluffo H, Acarin L, Faiz M, Castellano B, Gonzalez B: Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury. J Neuroinflammation. 2005, 2 (1): 12-10.1186/1742-2094-2-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Chan PH, Yang GY, Chen SF, Carlson E, Epstein CJ: Cold-induced brain edema and infarction are reduced in transgenic mice overexpressing CuZn-superoxide dismutase. Ann Neurol. 1991, 29 (5): 482-486. 10.1002/ana.410290506.View ArticlePubMedGoogle Scholar
- Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H: Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994, 25 (1): 165-170.View ArticlePubMedGoogle Scholar
- Mikawa S, Kinouchi H, Kamii H, Gobbel GT, Chen SF, Carlson E, Epstein CJ, Chan PH: Attenuation of acute and chronic damage following traumatic brain injury in copper, zinc-superoxide dismutase transgenic mice. J Neurosurg. 1996, 85 (5): 885-891.View ArticlePubMedGoogle Scholar
- Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K, Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH: Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci. 1997, 17 (11): 4180-4189.PubMedGoogle Scholar
- Sheng H, Brady TC, Pearlstein RD, Crapo JD, Warner DS: Extracellular superoxide dismutase deficiency worsens outcome from focal cerebral ischemia in the mouse. Neurosci Lett. 1999, 267 (1): 13-16. 10.1016/S0304-3940(99)00316-X.View ArticlePubMedGoogle Scholar
- Berger R, Garnier Y: Pathophysiology of perinatal brain damage. Brain Res Brain Res Rev. 1999, 30 (2): 107-134. 10.1016/S0165-0173(99)00009-0.View ArticlePubMedGoogle Scholar
- Ferriero DM: Neonatal brain injury. N Engl J Med. 2004, 351 (19): 1985-1995. 10.1056/NEJMra041996.View ArticlePubMedGoogle Scholar
- Ikonomidou C, Mosinger JL, Salles KS, Labruyere J, Olney JW: Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methyl-aspartate neurotoxicity. J Neurosci. 1989, 9 (8): 2809-2818.PubMedGoogle Scholar
- Olney JW: Excitotoxin-mediated neuron death in youth and old age. Prog Brain Res. 1990, 86: 37-51.View ArticlePubMedGoogle Scholar
- Acarin L, Peluffo H, Gonzalez B, Castellano B: Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain. J Neurosci Res. 2002, 68 (6): 745-754. 10.1002/jnr.10261.View ArticlePubMedGoogle Scholar
- Acarin L, Peluffo H, Barbeito L, Castellano B, Gonzalez B: Astroglial nitration after postnatal excitotoxic damage: correlation with nitric oxide sources, cytoskeletal, apoptotic and antioxidant proteins. J Neurotrauma. 2005, 22 (1): 189-200. 10.1089/neu.2005.22.189.View ArticlePubMedGoogle Scholar
- Ditelberg JS, Sheldon RA, Epstein CJ, Ferriero DM: Brain injury after perinatal hypoxia-ischemia is exacerbated in copper/zinc superoxide dismutase transgenic mice. Pediatr Res. 1996, 39 (2): 204-208.View ArticlePubMedGoogle Scholar
- Fullerton HJ, Ditelberg JS, Chen SF, Sarco DP, Chan PH, Epstein CJ, Ferriero DM: Copper/zinc superoxide dismutase transgenic brain accumulates hydrogen peroxide after perinatal hypoxia ischemia. Ann Neurol. 1998, 44 (3): 357-364. 10.1002/ana.410440311.View ArticlePubMedGoogle Scholar
- Shimizu K, Rajapakse N, Horiguchi T, Payne RM, Busija DW: Neuroprotection against hypoxia-ischemia in neonatal rat brain by novel superoxide dismutase mimetics. Neurosci Lett. 2003, 346 (1-2): 41-44. 10.1016/S0304-3940(03)00558-5.View ArticlePubMedGoogle Scholar
- Fan P, Yamauchi T, Noble LJ, Ferriero DM: Age-dependent differences in glutathione peroxidase activity after traumatic brain injury. J Neurotrauma. 2003, 20 (5): 437-445. 10.1089/089771503765355513.View ArticlePubMedGoogle Scholar
- Palmer C, Menzies SL, Roberts RL, Pavlick G, Connor JR: Changes in iron histochemistry after hypoxic-ischemic brain injury in the neonatal rat. J Neurosci Res. 1999, 56 (1): 60-71. 10.1002/(SICI)1097-4547(19990401)56:1<60::AID-JNR8>3.0.CO;2-A.View ArticlePubMedGoogle Scholar
- Kondo Y, Ogawa N, Asanuma M, Ota Z, Mori A: Regional differences in late-onset iron deposition, ferritin, transferrin, astrocyte proliferation, and microglial activation after transient forebrain ischemia in rat brain. J Cereb Blood Flow Metab. 1995, 15 (2): 216-226.View ArticlePubMedGoogle Scholar
- Ebadi M: Biochemical alteration of a metallothionein-like protein in developing rat brain. Biol Trace Elem Res. 1986, 11: 117-128.View ArticlePubMedGoogle Scholar
- Nishimura N, Nishimura H, Ghaffar A, Tohyama C: Localization of metallothionein in the brain of rat and mouse. J Histochem Cytochem. 1992, 40 (2): 309-315.View ArticlePubMedGoogle Scholar
- McDonald JW, Silverstein FS, Johnston MV: Neurotoxicity of N-methyl-D-aspartate is markedly enhanced in developing rat central nervous system. Brain Res. 1988, 459 (1): 200-203. 10.1016/0006-8993(88)90306-X.View ArticlePubMedGoogle Scholar
- Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J: NMDA-dependent superoxide production and neurotoxicity. Nature. 1993, 364 (6437): 535-537. 10.1038/364535a0.View ArticlePubMedGoogle Scholar
- Dugan LL, Sensi SL, Canzoniero LM, Handran SD, Rothman SM, Lin TS, Goldberg MP, Choi DW: Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci. 1995, 15 (10): 6377-6388.PubMedGoogle Scholar
- Peluffo H, Aris A, Acarin L, Gonzalez B, Villaverde A, Castellano B: Nonviral gene delivery to the central nervous system based on a novel integrin-targeting multifunctional protein. Hum Gene Ther. 2003, 14 (13): 1215-1223. 10.1089/104303403767740759.View ArticlePubMedGoogle Scholar
- Xiang JJ, Tang JQ, Zhu SG, Nie XM, Lu HB, Shen SR, Li XL, Tang K, Zhou M, Li GY: IONP-PLL: a novel non-viral vector for efficient gene delivery. J Gene Med. 2003, 5 (9): 803-817. 10.1002/jgm.419.View ArticlePubMedGoogle Scholar
- Wang J, Zhang PC, Lu HF, Ma N, Wang S, Mao HQ, Leong KW: New polyphosphoramidate with a spermidine side chain as a gene carrier. J Control Release. 2002, 83 (1): 157-168. 10.1016/S0168-3659(02)00180-3.View ArticlePubMedGoogle Scholar
- Shi N, Pardridge WM: Noninvasive gene targeting to the brain. Proc Natl Acad Sci U S A. 2000, 97 (13): 7567-7572. 10.1073/pnas.130187497.PubMed CentralView ArticlePubMedGoogle Scholar
- Bessis N, GarciaCozar FJ, Boissier MC: Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 2004, 11 Suppl 1: S10-7. 10.1038/sj.gt.3302364.View ArticlePubMedGoogle Scholar
- Maguire-Zeiss KA, Federoff HJ: Safety of viral vectors for neurological gene therapies. Curr Opin Mol Ther. 2004, 6 (5): 473-481.PubMedGoogle Scholar
- Aris A, Feliu JX, Knight A, Coutelle C, Villaverde A: Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression. Biotechnol Bioeng. 2000, 68 (6): 689-696. 10.1002/(SICI)1097-0290(20000620)68:6<689::AID-BIT13>3.0.CO;2-A.View ArticlePubMedGoogle Scholar
- Aris A, Villaverde A: Molecular organization of protein-DNA complexes for cell-targeted DNA delivery. Biochem Biophys Res Commun. 2000, 278 (2): 455-461. 10.1006/bbrc.2000.3824.View ArticlePubMedGoogle Scholar
- Aris A, Villaverde A: Engineering nuclear localization signals in modular protein vehicles for gene therapy. Biochem Biophys Res Commun. 2003, 304 (4): 625-631. 10.1016/S0006-291X(03)00644-2.View ArticlePubMedGoogle Scholar
- Acarin L, Gonzalez B, Castellano B, Castro AJ: Microglial response to N-methyl-D-aspartate-mediated excitotoxicity in the immature rat brain. J Comp Neurol. 1996, 367 (3): 361-374. 10.1002/(SICI)1096-9861(19960408)367:3<361::AID-CNE4>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Radi R: Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A. 2004, 101 (12): 4003-4008. 10.1073/pnas.0307446101.PubMed CentralView ArticlePubMedGoogle Scholar
- Johnston MV, Nakajima W, Hagberg H: Mechanisms of hypoxic neurodegeneration in the developing brain. Neuroscientist. 2002, 8 (3): 212-220.View ArticlePubMedGoogle Scholar
- Dirnagl U, Iadecola C, Moskowitz MA: Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999, 22 (9): 391-397. 10.1016/S0166-2236(99)01401-0.View ArticlePubMedGoogle Scholar
- Obrenovitch TP, Urenjak J: Is high extracellular glutamate the key to excitotoxicity in traumatic brain injury?. J Neurotrauma. 1997, 14 (10): 677-698.View ArticlePubMedGoogle Scholar
- Cotman CW: Apoptosis decision cascades and neuronal degeneration in Alzheimer's disease. Neurobiol Aging. 1998, 19 (1 Suppl): S29-32. 10.1016/S0197-4580(98)00042-6.View ArticlePubMedGoogle Scholar
- Jenner P, Olanow CW: Understanding cell death in Parkinson's disease. Ann Neurol. 1998, 44 (3 Suppl 1): S72-84.View ArticlePubMedGoogle Scholar
- Petersen A, Mani K, Brundin P: Recent advances on the pathogenesis of Huntington's disease. Exp Neurol. 1999, 157 (1): 1-18. 10.1006/exnr.1998.7006.View ArticlePubMedGoogle Scholar
- Pardo CA, Xu Z, Borchelt DR, Price DL, Sisodia SS, Cleveland DW: Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons. Proc Natl Acad Sci U S A. 1995, 92 (4): 954-958.PubMed CentralView ArticlePubMedGoogle Scholar
- Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990, 87 (4): 1620-1624.PubMed CentralView ArticlePubMedGoogle Scholar
- Radi R, Beckman JS, Bush KM, Freeman BA: Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem. 1991, 266 (7): 4244-4250.PubMedGoogle Scholar
- Chen Y, Chan PH, Swanson RA: Astrocytes overexpressing Cu,Zn superoxide dismutase have increased resistance to oxidative injury. Glia. 2001, 33 (4): 343-347. 10.1002/1098-1136(20010315)33:4<343::AID-GLIA1033>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Wang J, Ma JH, Giffard RG: Overexpression of copper/zinc superoxide dismutase decreases ischemia-like astrocyte injury. Free Radic Biol Med. 2005, 38 (8): 1112-1118. 10.1016/j.freeradbiomed.2005.01.010.View ArticlePubMedGoogle Scholar
- Cassina P, Peluffo H, Pehar M, Martinez-Palma L, Ressia A, Beckman JS, Estevez AG, Barbeito L: Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J Neurosci Res. 2002, 67 (1): 21-29. 10.1002/jnr.10107.View ArticlePubMedGoogle Scholar
- Estevez AG, Sampson JB, Zhuang YX, Spear N, Richardson GJ, Crow JP, Tarpey MM, Barbeito L, Beckman JS: Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor withdrawal. Free Radic Biol Med. 2000, 28 (3): 437-446. 10.1016/S0891-5849(99)00261-0.View ArticlePubMedGoogle Scholar
- Estevez AG, Spear N, Manuel SM, Radi R, Henderson CE, Barbeito L, Beckman JS: Nitric oxide and superoxide contribute to motor neuron apoptosis induced by trophic factor deprivation. J Neurosci. 1998, 18 (3): 923-931.PubMedGoogle Scholar
- Peluffo H, Shacka JJ, Ricart K, Bisig CG, Martinez-Palma L, Pritsch O, Kamaid A, Eiserich JP, Crow JP, Barbeito L, Estevez AG: Induction of motor neuron apoptosis by free 3-nitro-L-tyrosine. J Neurochem. 2004, 89 (3): 602-612. 10.1046/j.1471-4159.2004.02363.x.View ArticlePubMedGoogle Scholar
- Vergun O, Sobolevsky AI, Yelshansky MV, Keelan J, Khodorov BI, Duchen MR: Exploration of the role of reactive oxygen species in glutamate neurotoxicity in rat hippocampal neurones in culture. J Physiol. 2001, 531 (Pt 1): 147-163. 10.1111/j.1469-7793.2001.0147j.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Barkats M, Bemelmans AP, Geoffroy MC, Robert JJ, Loquet I, Horellou P, Revah F, Mallet J: An adenovirus encoding CuZnSOD protects cultured striatal neurones against glutamate toxicity. Neuroreport. 1996, 7 (2): 497-501.View ArticlePubMedGoogle Scholar
- Chan PH, Chu L, Chen SF, Carlson EJ, Epstein CJ: Reduced neurotoxicity in transgenic mice overexpressing human copper-zinc-superoxide dismutase. Stroke. 1990, 21 (11 Suppl): III80-2.PubMedGoogle Scholar
- Borg J, London J: Copper/zinc superoxide dismutase overexpression promotes survival of cortical neurons exposed to neurotoxins in vitro. J Neurosci Res. 2002, 70 (2): 180-189. 10.1002/jnr.10404.View ArticlePubMedGoogle Scholar
- Ying W, Anderson CM, Chen Y, Stein BA, Fahlman CS, Copin JC, Chan PH, Swanson RA: Differing effects of copper,zinc superoxide dismutase overexpression on neurotoxicity elicited by nitric oxide, reactive oxygen species, and excitotoxins. J Cereb Blood Flow Metab. 2000, 20 (2): 359-368. 10.1097/00004647-200002000-00018.View ArticlePubMedGoogle Scholar
- Dawson TM, Steiner JP, Dawson VL, Dinerman JL, Uhl GR, Snyder SH: Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci U S A. 1993, 90 (21): 9808-9812.PubMed CentralView ArticlePubMedGoogle Scholar
- Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, Loscalzo J, Singel DJ, Stamler JS: A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature. 1993, 364 (6438): 626-632. 10.1038/364626a0.View ArticlePubMedGoogle Scholar
- Gunasekar PG, Kanthasamy AG, Borowitz JL, Isom GE: NMDA receptor activation produces concurrent generation of nitric oxide and reactive oxygen species: implication for cell death. J Neurochem. 1995, 65 (5): 2016-2021.View ArticlePubMedGoogle Scholar
- Ceballos-Picot I, Nicole A, Clement M, Bourre JM, Sinet PM: Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase. Mutat Res. 1992, 275 (3-6): 281-293.View ArticlePubMedGoogle Scholar
- Jaarsma D, Haasdijk ED, Grashorn JA, Hawkins R, van Duijn W, Verspaget HW, London J, Holstege JC: Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol Dis. 2000, 7 (6 Pt B): 623-643. 10.1006/nbdi.2000.0299.View ArticlePubMedGoogle Scholar
- Dal Canto MC, Gurney ME: Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res. 1995, 676 (1): 25-40. 10.1016/0006-8993(95)00063-V.View ArticlePubMedGoogle Scholar
- Avraham KB, Schickler M, Sapoznikov D, Yarom R, Groner Y: Down's syndrome: abnormal neuromuscular junction in tongue of transgenic mice with elevated levels of human Cu/Zn-superoxide dismutase. Cell. 1988, 54 (6): 823-829. 10.1016/S0092-8674(88)91153-1.View ArticlePubMedGoogle Scholar
- Gahtan E, Auerbach JM, Groner Y, Segal M: Reversible impairment of long-term potentiation in transgenic Cu/Zn-SOD mice. Eur J Neurosci. 1998, 10 (2): 538-544. 10.1046/j.1460-9568.1998.00058.x.View ArticlePubMedGoogle Scholar
- Weinzierl M, Mautes AE, Lin Y, Noble LJ: Attenuated induction of heme oxygenase after intrathecal exposure to lysed blood in mice overexpressing superoxide dismutase. Neurosci Lett. 2003, 336 (1): 13-16. 10.1016/S0304-3940(02)01233-8.View ArticlePubMedGoogle Scholar
- Sheldon RA, Jiang X, Francisco C, Christen S, Vexler ZS, Tauber MG, Ferriero DM: Manipulation of antioxidant pathways in neonatal murine brain. Pediatr Res. 2004, 56 (4): 656-662. 10.1203/01.PDR.0000139413.27864.50.View ArticlePubMedGoogle Scholar
- He YY, Hsu CY, Ezrin AM, Miller MS: Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion. Am J Physiol. 1993, 265 (1 Pt 2): H252-6.PubMedGoogle Scholar
- Dumas TC, Sapolsky RM: Gene therapy against neurological insults: sparing neurons versus sparing function. Trends Neurosci. 2001, 24 (12): 695-700. 10.1016/S0166-2236(00)01956-1.View ArticlePubMedGoogle Scholar
- Zhang Y, Schlachetzki F, Zhang YF, Boado RJ, Pardridge WM: Normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism with intravenous nonviral gene therapy and a brain-specific promoter. Hum Gene Ther. 2004, 15 (4): 339-350. 10.1089/104303404322959498.View ArticlePubMedGoogle Scholar
- Aris A, Villaverde A: Modular protein engineering for non-viral gene therapy. Trends Biotechnol. 2004, 22 (7): 371-377. 10.1016/j.tibtech.2004.05.004.View ArticlePubMedGoogle Scholar
- Borsello T, Bressoud R, Mottier V, Gonzalez N, Gomez G, Clarke PG: Kainate-induced endocytosis in retinal amacrine cells. J Comp Neurol. 2003, 465 (2): 286-295. 10.1002/cne.10834.View ArticlePubMedGoogle Scholar
- Borsello T, Croquelois K, Hornung JP, Clarke PG: N-methyl-d-aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathway. Eur J Neurosci. 2003, 18 (3): 473-485. 10.1046/j.1460-9568.2003.02757.x.View ArticlePubMedGoogle Scholar
- Duque H, Baxt B: Foot-and-mouth disease virus receptors: comparison of bovine alpha(V) integrin utilization by type A and O viruses. J Virol. 2003, 77 (4): 2500-2511. 10.1128/JVI.77.4.2500-2511.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Gris D, Marsh DR, Oatway MA, Chen Y, Hamilton EF, Dekaban GA, Weaver LC: Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function. J Neurosci. 2004, 24 (16): 4043-4051. 10.1523/JNEUROSCI.5343-03.2004.View ArticlePubMedGoogle Scholar
- Qin J, Vinogradova O, Plow EF: Integrin bidirectional signaling: a molecular view. PLoS Biol. 2004, 2 (6): 726-729. 10.1371/journal.pbio.0020169.View ArticleGoogle Scholar
- McInnis R, Perkinson RA, Kuo BA, Norton PA: Unexpected nuclear localization of a chimeric b-galactosidase lacZ reporter gene product in mammalian cells. Biochem Mol Biol Int. 1995, 36: 483-490.PubMedGoogle Scholar
- Acarin L, Gonzalez B, Castro AJ, Castellano B: Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: microglial/macrophage response and major histocompatibility complex class I and II expression. Neuroscience. 1999, 89 (2): 549-565. 10.1016/S0306-4522(98)00331-5.View ArticlePubMedGoogle Scholar
- Marshall JF, Ungerstedt U: Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model. Eur J Pharmacol. 1977, 41 (4): 361-367. 10.1016/0014-2999(77)90256-4.View ArticlePubMedGoogle Scholar
- Schmued LC, Hopkins KJ: Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration. Toxicol Pathol. 2000, 28 (1): 91-99.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.