Ann Thorac Surg 2004;78:602-607
© 2004 The Society of Thoracic Surgeons
Original article: cardiovascular
MCI-186 reduces oxidative cellular damage and increases DNA repair function in the rabbit spinal cord after transient ischemia
Goro Takahashi, MDa,
Masahiro Sakurai, MD, PhDa*,
Koji Abe, MD, PhDc,
Yasuto Itoyama, MD, PhDb,
Koichi Tabayashi, MD, PhDa
a Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan
b Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan
c Department of Neurology, Okayama University Medical School, Okayama, Japan
Accepted for publication February 17, 2004.
* Address reprint requests to Dr Sakurai, Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
e-mail: sakuraim{at}mail.tains.tohoku.ac.jp
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Abstract
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BACKGROUND: Paraplegia is a serious complication of operations on the thoracic and thoracoabdominal aorta. To investigate the mechanism by which motor neurons are damaged during these operations, we have reported a rabbit model of spinal cord ischemia. We also tested whether a free radical scavenger MCI-186 that is useful for treating ischemic damage in the brain can protect against ischemic spinal cord damage.
METHODS: Fifteen minutes of ischemia was induced, then MCI-186 or vehicle was injected intravenously. Cell damage was analyzed by observing the function of the lower limbs and by counting the number of motor neurons. To investigate the mechanism by which MCI-186 prevents ischemic spinal cord damage, we observed the immunoreactivity of 8-hydroxy-2'-deoxyguanosine as an oxidative DNA damage marker and redox effector as a DNA repair marker.
RESULTS: In sham control, 8-hydroxy-2'-deoxyguanosine was not observed, and the nuclear expression of redox effector was observed. In vehicle injection group (group I), the nuclear expression of 8-hydroxy-2'-deoxyguanosine was observed at 1 and 2 days after reperfusion. The nuclear expression of redox effector was observed at 8 hours and 1 day, and disappeared at 2 days after transient ischemia. In MCI-186 injection group (group M), the nuclear expression of 8-hydroxy-2'-deoxyguanosine was not observed, and redox effector was observed at 8 hours and 1 and 2 days.
CONCLUSIONS: These results suggest that redox effector decreased in motor neurons after transient ischemia and this reduction preceded oxidative DNA damage. MCI-186 works as a radical scavenger and reduced oxidative DNA damage, so redox effector did not disappear. MCI-186 could be a strong candidate for a use as a therapeutic agent in the treatment of ischemic spinal cord injury.
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Introduction
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Spinal cord injury after a successful operation on the thoracic aorta is a disastrous and unpredictable complication in humans. In an attempt to prevent this complication, various methods of spinal cord protection have been suggested [1–3], including temporary shunts, partial bypass, and systemic or regional hypothermia. Regardless of the surgical technique or method of spinal cord protection used, no method has been developed that totally prevents the development of paraplegia [4]. The reported incidence of paraplegia ranges from 5% to 15% in operations on the thoracic aorta [5,]. The mechanism of spinal cord injury during operations on the thoracic aorta is thought to relate primarily to tissue ischemia. Ischemia can occur because of permanent exclusion of the essential intercostal arterial blood supply to the spinal cord or by temporary interruption of the spinal cord blood flow [6, 7]. Spinal motor neurons are thought to be more vulnerable to ischemia than dorsal horn neurons. However, the exact mechanism is not fully understood. To evaluate the exact mechanism of such vulnerability of spinal motor neurons by ischemia, we attempted to make a reproducible model for spinal cord ischemia and statistically analyzed cell damage [8, 9].
Under conditions of severe oxidative stress, the C-8 position of 2'-deoxyguanosine, a constituent of DNA, is hydroxylated and 8-hydroxy-2'-deoxyguanosine (8-OHdG) is produced [10]. Ischemic insults produce reactive oxygen species , such as hydroxyl radical, superoxide anion, and peroxynitrite. Interaction between DNA and reactive oxygen species produces DNA strand breaks and base modification, which are frequently assessed by measurement of the nucleoside 8-OHdG levels [11]. Thus, it is currently considered that 8-OHdG is one of the best markers of the oxidative DNA damage.
Apurinic/apyrimidinic endonuclease (APE), or redox effector factor (Ref-1), is a multifunctional enzyme involved in the base excision DNA repair of apurinic and apyrimidinic sites in DNA, which is a common form of DNA damage after oxidative stress [12]. The base excision repair pathway is initiated by removal of the damaged base by a DNA glycosylase, which generates an apurinic or apyrimidinic site. Thereafter, APE/Ref-1 cleaves the apyrimidinic site to 5'-phosphodeoxyribose, thus generating a free 3'-hydroxyl required for DNA polymerase to fill the resulting gap [13].
We propose that MCI-186 protects the DNA from damage because of the reduction of nitric oxide, superoxide, and peroxynitrite, and Ref-1 was not exhausted.
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Material and methods
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Thirty-six male domesticated white rabbits (Funakoshi, Japan), weighing 2 to 3 kg, were divided into three groups: sham control group, transient ischemia and vehicle treatment group (group I), and transient ischemia and treatment with MCI-186 group (group M). All rabbits were allowed free access to food and water before and after the procedure. Anesthesia was induced with intramuscular administration of ketamine at a dose of 50 mg/kg and maintained with 2% halothane inhalation in oxygen. A 4F Fogarty catheter (Baxter, Germany) was inserted through the right femoral artery and advanced 15 cm forward into the abdominal aorta. Our preliminary experiments in 10 animals had already confirmed that the balloon in the distal end of the catheter should be positioned 0.5 to 1.5 cm distal to the left renal artery. The catheter was immediately removed without injection or balloon inflation in the sham control animals. Our previous experiments confirmed that 15 minutes of transient spinal cord ischemia resulted in selective motor neuron death, which might be a part of the apoptotic change [9]. We applied this ischemic model to the analysis of the effect of MCI-186. MCI-186 (3 mg/kg) or vehicle was administrated intravenously after 30 minutes of reperfusion.
Later the rabbits were killed at 8 hours or 1, 2, and 7 days after reperfusion (n = 3 for each group at 1 and 2 days, and n = 6 for each group at 7 days after reperfusion). Immediately after the animals died, the spinal cord was quickly removed with the plunger of a 1-mL syringe [14]. All sample were frozen in powdered dry ice and stored at –80°C until use. Then the spinal cords were cut transversely at approximately the L2 or L3 level and, finally, mounted on a glass slide. In the experiment, rabbits were treated in accordance with the Declaration of Helsinki and the "Guidelines for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Also, both the experimental and animal care protocols were approved by the Animal Care Committee of the Tohoku University School of Medicine.
Mouse monoclonal antibody against 8-OHdG (MOG-020; JICA, Shizuoka, Japan), at a concentration of 7.0 µg/mL in 2% normal horse serum, and anti-APE/Ref-1 polyclonal antibody (Novus Biologicals, Littleton, CO), at a dilution of 1:100, were used for immunohistochemical study. The specificity of these antibodies had been established elsewhere [11, 13].
For immunohistochemical analysis, the sections were fixed for 10 minutes in ice-cold acetone, air-dried, and rinsed in 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl (pH 7.4). After being blocked with 10% normal horse serum for 2 hours, the slides were washed and incubated overnight at 4°C with each antibody. Endogenous peroxidase was blocked for 20 minutes with 0.3% hydrogen peroxide and 10% methanol. Then sections were washed and incubated for 3 hours with biotinylated anti-mouse immunoglobulin G (1:200 dilution; BA-2000, Vector Laboratories, Burlingame, CA) in the buffer, followed by incubation for 30 minutes with the avidin-biotin-peroxidase complex. Staining was developed with 3,3'-diaminobenzidine tetrahydrochloride (0.5 mg/mL in 50 mmol/L Tris-HCl buffer, pH 7.4) in the presence of hydrogen peroxide.
The results were expressed as the mean ± standard deviation. The Mann-Whitney U test or paired Student's t test was used for comparison of normal data.
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Results
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Neurologic assessment
Neurologic function was assessed before the animals were put to death 8 hours, 1 day, 2 days, or 7 days (n = 3 at 8 hours and 1 and 2 days, n = 6 at sham and 7 days) after reperfusion. Animals were classified by a 5-point scale devised by Johnson and associates [15] as follows: 0 = hind-limb paralysis; 1 = severe paraparesis; 2 = functional movement, no hop; 3 = ataxia, uncoordinated hop; 4 = minimal ataxia; 5 = normal function. Two individuals without knowledge of the treatment independently graded neurologic function. The neurologic scores are summarized in Table 1. The Mann-Whitney U test was used to compare the neurologic scores.
Histologic study
The numbers of intact motor neurons are summarized in Table 2. In sham-operated control animals, the spinal cord was intact, with many large motor neurons in the anterior horn. However, in the spinal cord at 7 days after restoration of blood flow in group I, more than 70% of motor neurons were damaged. Seven days after reperfusion in group M, there was less evidence of damaged motor neurons. The number of intact motor neurons in group I was significantly smaller than that in group M (p = 0.004; Fig 1).
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Fig 1. Hematoxylin & eosin staining in spinal cord specimens obtained from sham-operated animal (a) and at 7 days from animal in transient ischemia and vehicle treatment group (group I; b) and from animal in transient ischemia and treatment with MCI-186 group (group M; c). Bar represents 100 µm.
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Immunohistochemical study
To detect the oxidative DNA damage, we examined production of 8-OHdG in the motor neurons of the anterior horns. There was no detectable 8-OHdG in the sham controls (Fig 2a). At 1 and 2 days after reperfusion, however, immunoreactivity for 8-OHdG became strongly positive in the motor neurons in group I (Fig 2c, 2 days). However, there was no detection of the expression of 8-OHdG in group M (Figs 2e, 2f, 2g). The number of motor neurons in the anterior horns that were stained with 8-OHdG in group I is significantly less than that in group M. It was demonstrated that nuclei were most densely stained.
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Fig 2. Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine in spinal cord specimens obtained from sham-operated animal (a), at 8 hours (b), 1 day (c), and 2 days (d) after ischemia from animal in transient ischemia and vehicle treatment group (group I), and at 8 hours (e), 1 day (f), and 2 days (g) after ischemia from animal in transient ischemia and treatment with MCI-186 group (group M). The 8-hydroxy-2'-deoxyguanosine–positive cells (arrowheads) in group I were observed at 1 and 2 days. No 8-hydroxy-2'-deoxyguanosine–positive cells were observed in group M. Bar represents 100 µm.
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In sham-operated animals, APE/Ref-1 immunoreactivity was located in the nuclei (Fig 3a). It was widely distributed in the spinal cord, but was most prominent in the anterior horns. At 8 hours after transient ischemia, there was a clear reduction in nuclear APE/Ref-1 immunoreactivity in the anterior horns of the spinal cord (Fig 3b). The loss of APE/Ref-1 immunoreactivity in these regions remained during the observation period. Some cells showed increased staining in regions adjacent to APE/Ref-1 loss.
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Fig 3. Immunohistochemical staining for redox effector factor (Ref-1) in spinal cord obtained from sham-operated animal (a), at 8 hours (b), 1 day (c), and 2 days (d) after ischemia from animal in transient ischemia and vehicle treatment group (group I), and at 8 hours (e), 1 day (f), and 2 days (g) after ischemia from animal in transient ischemia and treatment with MCI-186 group (group M). Redox effector factor immunoreactivity (arrowheads) in group I was observed until 1 day; however, it disappeared at 2 days. Redox effector factor immunoreactivity in group M was clearly observed until 2 days. Bar represents 40 µm.
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At 8 hours after reperfusion in group M, there was no remarkable change in the immunoreactivity to APE/Ref-1 (Fig 3e). The number of APE/Ref-1–positive motor neurons was more increased in group M than in group I. However, the number of alive motor neurons is larger in group M than in group I. Therefore, we compared the ratio of APE/Ref-1–positive motor neurons to alive motor neurons in group I to that in group M. The ratio in group M was not different from that in group I.
We showed that MCI-186 reduced the expression of 8-OHdG, but did not increase the expression of APE/Ref-1.
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Comment
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We have already demonstrated delayed selective motor neuron death in lumber lesions of the rabbit spinal cord with a reproducible ischemia model, and the death might reflect an apoptotic change [16]. We used MCI-186 to potentially protect the ischemic and postischemic spinal cord injury.
There was a report that MCI-186 markedly attenuated the ischemic and postischemic brain swelling [17]. MCI-186 was shown to inhibit a peroxidative mechanism in vitro, which has been implicated as a detrimental factor in ischemic and postischemic cell damage [18]. There are two pathways generating peroxidative reactions in damaged brain as well as in any tissue: one is autooxidation (nonenzymatic peroxidation) and the other is enzyme-processing peroxidation [19]. Cell damage induced by nonenzymatic peroxidation within an in vitro system is suggested to be attributable to iron, superoxide dismutase, hydrogen peroxide, and lipoxygenase products from arachidonic acid (enzyme-processing peroxidation) [20].
It was widely accepted that in the middle cerebral artery occlusion model, a marked increase in water content at the ischemic area may be attributable to a sufficient water supply resulting from reperfusion corresponding to the potential of tissue swelling [17]. Furthermore, marked generation of superoxide derived from arachidonate metabolism may also cause a deterioration of cell damage and may result in the aggravation of brain edema during reperfusion [21]. Introduction of arachidonate or a potent initiator of free radical species induces brain edema in vivo [22].
Severe oxidative stress causes various kinds of damage in cells, but the damage on DNA is particular relevance [23]. 8-Hydroxy-2'-deoxyguanosine is considered one of the DNA oxidation byproducts. It is generated in cells with oxidative damage but not in those with nonoxidative damage [24]. Because 8-OHdG induces conversion of guanine-cytosine to thymine-adenine, 8-OHdG itself might result in impairment of normal cell function by means of this conversion in cells that evaded acute lethal cellular injury [25]. We show in this report that the expression of 8-OHdG disappeared during 2 days after reperfusion with injection of MCI-186. MCI-186 acts as a radical scavenger and neutralizes reactive oxygen species produced by ischemic insults.
In the study of DNA repair mechanisms, Ref-1 is drawing particular attention because of its critical role in the redox regulation of DNA-binding activity of activator protein-1 family members, such as Fos and Jun transcription factors [26], which are also considered to be associated with the pathogenesis of cerebral ischemia [27]. However, recent in vivo studies showed a lack of correlation between Ref-1 protein levels and expression of inducible transcription factor c-Fos and c-Jun, suggesting that Ref-1 protein is more likely to be involved in the repair of spontaneous DNA damage than posttranslational modification [28]. We show in this report that with injection of MCI-186, nuclear expression of Ref-1 exists until 2 days after reperfusion. In group I, Ref-1 disappears at 2 days after transient ischemia. Levels of Ref-1 are dependent in inverse proportion to DNA damage. Therefore, MCI-186 was believed to reduce the DNA damage and prolong Ref-1.
After 15 minutes of ischemia of the spinal cord in rabbits, the expression of neural nitric oxide synthase, which is a cytotoxic factor and causes motor neuron death, was increased, and, thus, too much nitric oxide and peroxynitrite may be produced [29]. We suspect that nitric oxide, superoxide, and peroxynitrite will damage the DNA of motor neurons and contribute to the cell death with transient ischemia. However, there is no evidence whether nitric oxide, superoxide, and peroxynitrite will actually attack motor neuron cells and cause cell death. We show in this report that MCI-186 reduces the oxidative DNA damage of motor neurons and does not increase the expression of Ref-1, a DNA repair marker.
Recently, the protein levels of Ref-1 were shown to progressively decrease during the presymptomatic stage in a transgenic mouse model of amyotrophic lateral sclerosis [30]. In the present study we show that Ref-1 decreased at 1 day after transient ischemia and disappeared at 2 days. Motor neuron death after transient ischemia is thought to be an apoptotic change. MCI-186 prolongs Ref-1 and may prevent apoptotic changes in motor neurons. The mechanism of motor neuron death in the spinal cord after transient ischemia may be similar to that of familial amyotrophic lateral sclerosis. MCI-186 could become a strong candidate for use as a therapeutic agent in the treatment of ischemic spinal cord injury in the near future.
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References
|
|---|
- Symbas P.N., Pfaender L.M., Drucker M.H., Lester J.L., Gravaanis M.B., Zacharopoulos L. Cross-clamping of the descending aorta: hemodynamic and neurohumoral effects. J Thoracic Cardiovasc Surg 1983;85:300-305.[Medline]
- Svensson L.G., Rickards E., Coull A., Roger G., Fimmel C.J., Hinder R.A. Relationship of spinal cord blood flow to vascular anatomy during thoracic aortic cross-clamping and shunting. J Thorac Cardiovasc Surg 1986;91:71-78.[Abstract]
- Tabayashi K., Niibori K., Konno H., Mohri H. Protection from postischemic spinal cord injury by perfusion cooling of the epidural space. Ann Thorac Surg 1993;56:494-498.[Abstract/Free Full Text]
- Grossi E.A., Krieger K.H., Cunningham J.N., Jr, Culliford A.T., Nathan I.M., Spencer F.C. Venoarterial bypass: a technique for spinal cord protection. J Thorac Cardiovasc Surg 1985;89:228-234.[Abstract]
- Cambria R.P., Clouse W.D., Davidson J.K., Dunn P.F., Corey M., Dorer D. Thoracoabdominal aneurysm repair. Results with 337 operations performed over a 15-year interval. Ann Surg 2002;236:471-479.[Medline]
- Kats N.M., Blackstone E.H., Kirklin J.W., Karp R.B. Incremental risk factors for spinal cord injury following operation for acute aortic transection. J Thorac Cardiovasc Surg 1981;81:669-674.[Abstract]
- Livesay J.J., Cooley D.A., Ventemiglia R.A., et al. Surgical experience in descending thoracic aneurysmectomy with and without adjuncts to avoid ischemia. Ann Thorac Surg 1985;39:37-46.[Abstract/Free Full Text]
- Sakurai M., Aoki M., Abe K., Sadahiro M., Tabayashi K. Selective motor neuron death and heat shock protein induction after spinal cord ischemia in rabbits. J Thorac Cardiovasc Surg 1997;113:159-164.[Abstract/Free Full Text]
- Motoyoshi N., Sakurai M., Hayashi T., et al. Establishment of a focal cooling model against spinal cord ischemia representing prolonged induction of heat shock protein. J Thorac Cardiovasc Surg 2001;122:351-357.[Abstract/Free Full Text]
- Sakurai M., Nagata T., Abe K., Horinouchi T., Itoyama Y., Tabayashi K. Oxidative DNA damage and reduction of redox factor-1 expression after transient ischemia of spinal cord in rabbits. J Vasc Surg 2003;37:446-452.[Medline]
- Won M.H., Kang T.-C., Jeon G.-S., Kim D.-Y. Immunohistochemical detection of oxidative DNA damage induced by ischemia-reperfusion insults in gerbil hippocampus in vivo. Brain Res 1999;836:70-78.[Medline]
- Nngano I, Murakami T, Manabe Y, Abe K. Early decrease of survival factors and DNA repair enzyme in spinal motor neurons of presymptomatic transgenic mice that express a mutant SOD-1 gene. Life Sci 72:541–8
- Lewen A., Sugawara T., Gasche Y., Fujiyama M., Chan P.H. Oxidative cellular damage and reduction of APE/Ref-1 expression after experimental traumatic brain injury. Neurobiol Dis 2001;8:380-390.[Medline]
- Hayashi T., Sakurai M., Itoyama Y., Abe K. Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion. Brain Res 1999;832:159-163.[Medline]
- Johnson SH, Kraimer JM, Graeber GM. Effect of flunarizine on neurological recovery and spinal cord blood flow in experimental spinal cord ischemia in rabbits. Stroke 1993;24:1547–53
- Sakurai M., Fukuyama N., Takizawa S., et al. Inductions of 3-L-nitrotyrosine in motor neurons after transient spinal cord ischemia in rabbits. J Cereb Blood Flow Metab 1998;18:1233-1238.[Medline]
- Abe K., Yuki S., Kogure K. Strong attenuation oh ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988;19:480-485.[Abstract/Free Full Text]
- Flamm E.S., Demopoulos H.B., Seligman M.L., Poser R.G., Ransohoff J. Free radicals in cerebral ischemia. Stroke 1978;9:445-447.[Abstract/Free Full Text]
- Yamamoto Y., Kuwahara T., Watanabe K., Watanabe K. Antioxidant activity of 3-methyl-1-phenyl-2-pyrazolin-5-one. Redox Rep 1996;2:333-338.
- Abe K., Kogure K., Arai H., Nakano M. Ascorbate induced lipid peroxidation results in loss of receptor binding in Tris, but not in phosphate, buffer. Biochem Int 1985;11:341-348.[Medline]
- Kukreja R.C., Kontos H.A., Hess M.L., Ellis E.F. PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH. Circ Res 1986;59:612-619.[Abstract/Free Full Text]
- Chan P.H., Fishman R.A., Caronna J., Schmidley J.W., Priolean G., Lee J. Induction of brain edema following intracerebral injection of arachidonic acid. Ann Neurol 1983;13:625-632.[Medline]
- Mecocci P., Polidori M.C., Ingegni T., et al. Oxidative damage to DNA in lymphocytes from AD patients. Neurology 1998;51:1014-1017.[Abstract/Free Full Text]
- Toyokuni S., Tanaka Y., Hattori Y., et al. Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 1997;76:365-374.[Medline]
- Xanthoudakis S., Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J 1992;11:653-665.[Medline]
- Hirota K., Matsui M., Iwata S., Nishiyama A., Mori K., Yodoi J. AP-1 transcription activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci USA 1997;94:3663-3668.
- Kissling M., Gass P. Stimulus-transcription coupling in focal cerebral ischemia. Brain Pathol 1994;4:77-83.[Medline]
- Ono Y., Watanabe M., Inoue Y., et al. Developmental expression of APEX nuclease, a multifunctional DNA repair enzyme, in mouse brains. Dev Brain Res 1995;86:1-6.[Medline]
- Watanabe M., Sakurai M., Abe K., et al. Inductions of Cu/Zn superoxide dismutase- and nitric oxide synthase-like immunoreactivities in rabbit spinal cord after transient ischemia. Brain Res 1996;732:69-74.[Medline]
- Manabe Y., Warita H., Murakami T., et al. Early decrease of redox factor-1 in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene. Brain Res 2001;915:104-107.[Medline]
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Abstract
Introduction
