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• W2170146710 abstract "Paraplegia resulting from ischemia is a catastrophic complication of thoracoabdominal aortic surgery. The current study was designed to investigate the effects of diazoxide (DZ) on mitochondrial structure, neurological function, DNA damage-repair, and apoptosis in spinal cord ischemia-reperfusion injury. Rabbits were subjected to 30 minutes of spinal cord ischemia and reperfusion (1 hour) with or without diazoxide (n = 6 in each group) by clamping and releasing the infrarenal aorta. The neurological functional score was significantly improved in the DZ-treated ischemia-reperfusion injury group. Electron microscopic studies demonstrated that mitochondrial damage in the spinal cord after injury was significantly reduced by DZ. Mitochondrial superoxide and hydrogen peroxide levels were also markedly decreased in the DZ-treated injury group compared with the untreated group. DZ decreased levels of the oxidative DNA damage product 8-oxoG and increased levels of the DNA repair enzyme OGG-1. Furthermore, DZ inhibited apoptosis via caspase-dependent and -independent pathways. These studies indicate for the first time that the mitochondrial K-ATP channel opener diazoxide improves neurological function after spinal cord ischemia and reperfusion by diminishing levels of reactive oxygen species, decreasing DNA oxidative damage, and inhibiting caspase-dependent and -independent apoptotic pathways while preserving mitochondrial structure. Paraplegia resulting from ischemia is a catastrophic complication of thoracoabdominal aortic surgery. The current study was designed to investigate the effects of diazoxide (DZ) on mitochondrial structure, neurological function, DNA damage-repair, and apoptosis in spinal cord ischemia-reperfusion injury. Rabbits were subjected to 30 minutes of spinal cord ischemia and reperfusion (1 hour) with or without diazoxide (n = 6 in each group) by clamping and releasing the infrarenal aorta. The neurological functional score was significantly improved in the DZ-treated ischemia-reperfusion injury group. Electron microscopic studies demonstrated that mitochondrial damage in the spinal cord after injury was significantly reduced by DZ. Mitochondrial superoxide and hydrogen peroxide levels were also markedly decreased in the DZ-treated injury group compared with the untreated group. DZ decreased levels of the oxidative DNA damage product 8-oxoG and increased levels of the DNA repair enzyme OGG-1. Furthermore, DZ inhibited apoptosis via caspase-dependent and -independent pathways. These studies indicate for the first time that the mitochondrial K-ATP channel opener diazoxide improves neurological function after spinal cord ischemia and reperfusion by diminishing levels of reactive oxygen species, decreasing DNA oxidative damage, and inhibiting caspase-dependent and -independent apoptotic pathways while preserving mitochondrial structure. Paraplegia may complicate repairs of extensive thoracabdominal aortic aneurysms as a result of ischemic injury to the spinal cord that results from interruption of intercostal and lumbar arterial circulation.1Acher CW Wynn MM Hoch JR Popic P Archibald J Turnipseed WD Combined use of cerebral spinal fluid drainage and naloxone reduces the risk of paraplegia in thoracoabdominal aneurysm repair.J Vasc Surg. 1994; 19: 236-246Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 2Cambria RP Clouse WD Davison JK Dunn PF Corey M Dorer D Thoracoabdominal aneurysm repair: results with 337 operations performed over a 15-year interval.Ann Surg. 2002; 236: 471-479Crossref PubMed Scopus (313) Google Scholar, 3Cambria RP Davison JK Carter C Brewster DC Chang YC Clark KA Atamian S Epidural cooling for spinal cord protection during thoracoabdominal aneurysm repair: a five-year experience.J Vasc Surg. 2000; 31: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4Coselli JS LeMaire SA Left heart bypass reduces paraplegia rates after thoracoabdominal aortic aneurysm repair.Ann Thor Surg. 1999; 67: 1931-1934Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 5de Haan P Pharmacologic adjuncts to protect the spinal cord during transient ischemia.Semin Vasc Surg. 2000; 13: 264-271Abstract Full Text PDF PubMed Google Scholar Clinical adjuncts designed to reduce ischemia times, monitor neurological function, and allow for swelling of the spinal cord postoperatively have improved outcomes but do not eliminate the problem.6Jacobs MJ Meylaerts SA de Haan P de Mol BA Kalkman CJ Strategies to prevent neurologic deficit based on motor-evoked potentials in type I and II thoracoabdominal aortic aneurysm repair.J Vasc Surg. 1999; 29: 48-57Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Kouchoukos NT Masetti P Rokkas CK Murphy SF Hypothermic cardiopulmonary bypass and circulatory arrest for operations on the descending thoracic and thoracoabdominal aorta.Ann Thorac Surg. 2002; 74: S1885-S1887Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Safi HJ Bartoli S Hess KR Shenaq SS Viets JR Butt GR Sheinbaum R Doerr HK Maulsby R Rivera VM Neurologic deficit in patients at high risk with thoracoabdominal aortic aneurysms: the role of cerebral spinal fluid drainage and distal aortic perfusion.J Vasc Surg. 1994; 20: 434-444Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 9Safi HJ Miller III, CC Carr C Iliopoulos DC Dorsay DA Baldwin JC Importance of intercostal artery reattachment during thoracoabdominal aortic aneurysm repair.J Vasc Surg. 1998; 27: 58-66Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 10Safi HJ Campbell MP Miller III, CC Iliopoulos DC Khoynezhad A Letsou GV Asimacopoulos PJ Cerebral spinal fluid drainage and distal aortic perfusion decrease the incidence of neurological deficit: the results of 343 descending and thoracoabdominal aortic aneurysm repairs.Eur J Vasc Endovasc Surg. 1997; 14: 118-124Abstract Full Text PDF PubMed Scopus (96) Google Scholar Whereas complete postoperative neurological deficits were the rule in the past, today vascular surgeons are encountering more delayed and partial neurological deficits as a result of refinements in surgical technique. These types of neurological deficits result from ischemia and reperfusion injury and may potentially be ameliorated by pharmacological manipulation of this process. For this reason, our interest has centered on the basic mechanisms of injury of nervous tissue caused by ischemia and reperfusion.Important mechanisms in ischemia-reperfusion injury in neural tissue include glutamate receptor-mediated excitotoxicity, generation of reactive oxygen species (ROS), calcium influx, loss of membrane potential, mitochondrial failure, and apoptosis.11Dirnagl U Iadecola C Moskowitz MA Pathobiology of ischaemic stroke: an integrated view.Trends Neurosci. 1999; 22: 391-397Abstract Full Text Full Text PDF PubMed Scopus (3180) Google Scholar, 12Kristian T Siesjo BK Calcium-related damage in ischemia.Life Sci. 1996; 59: 357-367Crossref PubMed Scopus (187) Google Scholar, 13Lipton P Ischemic cell death in brain neurons.Physiol Rev. 1999; 79: 1431-1568Crossref PubMed Scopus (2561) Google Scholar Mitochondrial DNA is particularly sensitive to ischemia-reperfusion injury, and mitochondria play a central role in cell death signaling by releasing proteins that activate downstream phases of apoptosis. These proteins include cytochrome c, Smac, apoptosis-inducing factor (AIF), and endonuclease G.14Kaufmann SH Hengartner MO Programmed cell death: alive and well in the new millennium.Trends Cell Biol. 2001; 11: 526-534Abstract Full Text Full Text PDF PubMed Scopus (595) Google ScholarPrevention of mitochondrial failure improves cellular survival after an ischemic event. This phenomenon is seen in ischemic preconditioning, a process that involves mitochondrial K-ATP channels.15Gross GJ Auchampach JA Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs.Circ Res. 1992; 70: 223-233Crossref PubMed Scopus (932) Google Scholar Pharmacological agents that maintain mitochondrial K-ATP channels can prevent mitochondrial failure in a manner similar to that induced by ischemic preconditioning.16Garlid KD Paucek P Yarov-Yarovoy V Murray HN Darbenzio RB D'Alonzo AJ Lodge NJ Smith MA Grover GJ Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection.Circ Res. 1997; 81: 1072-1082Crossref PubMed Scopus (947) Google Scholar, 17Liu Y Sato T O'Rourke B Marban E Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection?.Circulation. 1998; 97: 2463-2469Crossref PubMed Scopus (731) Google Scholar, 18Liu Y Sato T Seharaseyon J Szewczyk A O'Rourke B Marban E Mitochondrial ATP-dependent potassium channels: viable candidate effectors of ischemic preconditioning.Ann NY Acad Sci. 1999; 874: 27-37Crossref PubMed Scopus (135) Google Scholar, 19Sato T Marban E The role of mitochondrial K(ATP) channels in cardioprotection.Basic Res Cardiol. 2000; 95: 285-289Crossref PubMed Scopus (66) Google Scholar Diazoxide (DZ), a specific opener of the mitochondrial membrane K-ATP channel, has been found to limit ischemia-reperfusion injury and apoptosis in a number of in vivo and in vitro models.16Garlid KD Paucek P Yarov-Yarovoy V Murray HN Darbenzio RB D'Alonzo AJ Lodge NJ Smith MA Grover GJ Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection.Circ Res. 1997; 81: 1072-1082Crossref PubMed Scopus (947) Google Scholar, 20Akao M Ohler A O'Rourke B Marban E Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.Circ Res. 2001; 88: 1267-1275Crossref PubMed Scopus (263) Google Scholar, 21Ichinose M Yonemochi H Sato T Saikawa T Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress.Am J Physiol Heart Circ Physiol. 2003; 284: H2235-H2241PubMed Google Scholar, 22Liu D Lu C Wan R Auyeung WW Mattson MP Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release.J Cereb Blood Flow Metab. 2002; 22: 431-443Crossref PubMed Scopus (202) Google Scholar, 23Takashi E Wang Y Ashraf M Activation of mitochondrial K(ATP) channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway.Circ Res. 1999; 85: 1146-1153Crossref PubMed Scopus (151) Google Scholar, 24Teshima Y Akao M Li RA Chong TH Baumgartner WA Johnston MV Marban E Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress.Stroke. 2003; 34: 1796-1802Crossref PubMed Scopus (117) Google ScholarIt has been shown in a rabbit model of spinal cord injury that diazoxide can ameliorate the effect of a temporary ischemic insult to the spinal cord.25Caparrelli DJ Cattaneo SM Bethea BT Shake JG Eberhart C Blue ME Marban E Johnston MV Baumgartner WA Gott VL Pharmacological preconditioning ameliorates neurological injury in a model of spinal cord ischemia.Ann Thorac Surg. 2002; 74: 838-844Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar In a previous study, we have demonstrated that a 30-minute period of aortic occlusion led to activation of caspase-3, oxi-dative damage to DNA, the activation and exhaustion of DNA repair enzymes, and apoptosis as demon-strated by transferase-mediated dUTP nick-end labeling (TUNEL).26Lin RX Roseborough G Dong YF Williams GM Wei CM DNA damage and repair system in spinal cord ischemia.J Vasc Surg. 2003; 37: 847-858Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar We hypothesized that mitochondrial K-ATP channel openers would prevent the accumulation of ROS and ameliorate the downstream effects that lead to apoptosis. The studies we report here document the beneficial clinical effect of diazoxide in this model through its effect on the production of reactive oxygen species in the mitochondria, the reduction of oxidative damage to DNA, the increased activity of the DNA repair enzyme OGG-1, the structural integrity of mitochondria, and the occurrence of apoptosis in spinal cord cells. Thus, diazoxide ameliorates the downstream apoptotic effects of mitochondrial injury while sustaining the activity of the DNA repair enzyme OGG-1 and the structural integrity of mitochondria.Materials and MethodsSurgical ProcedureIn a protocol approved by our Animal Care and Use Committee, New Zealand White rabbits weighing approximately 3 kg were administered a combination of intravenous medazalam (0.25 mg/kg), metomidine (0.05 mg/kg), atropine (0.04 mg/kg), and a bolus of propofol (2 mg/kg) to induce anesthesia and were sustained with a propofol drip (2 mg/kg/minute) through an ear vein. The contralateral auricular artery was cannulated, and the arterial pressure monitored by transducer. Sterile instruments and surgical techniques were used for all animals that were not sacrificed immediately postoperatively. The aorta was exposed through a small flank incision, the peritoneum was reflected anteriorly, and the aorta was identified and clamped just distal to the left renal artery. After 30 minutes of occlusion time, the clamp was removed, and the incision was closed in two layers. Animals were closely observed in the surgical suite. Postoperative analgesia was maintained with intramuscular injections of banamine (3 mg/kg).Animals receiving DZ were given an intravenous dose of 5 mg/kg 10 minutes before application of the infrarenal aortic clamp. Hypotension produced by DZ or by release of the aortic clamp was corrected by saline boluses in 50-ml increments. Mean arterial blood pressure was always maintained between 60 and 80 mmHg.The rabbits were sacrificed with lethal intravenous pentathol injections after 1 hour of reperfusion for biochemical studies or after 2 days for functional studies. For the biochemical studies, the aortic arch was rapidly exposed by sternotomy, the descending thoracic aorta was cannulated with intravenous tubing, and 1 L of saline at 4°C was infused. Outflow was achieved by opening the right atrium. Rapid laminectomy was performed from the cervical to the sacral vertebrae. Samples of the lumbar spinal cord were harvested at least 2 cm distal to the 12th rib to avoid contamination by nonischemic tissue. Samples of the thoracic cord were harvested above the 12th rib to serve as nonischemic controls. Samples were snap frozen, formalin fixed, or prepared for electron microscopy study as described. Spinal cord tissue (L4 level) was used for electron microscopy study and immunohistochemical sampling. The rest of the lumbar spinal cord was used for ROS generation study and Western blot analysis.Preparation of MitochondriaRabbit spinal cord mitochondria were isolated from the cortex of New Zealand White rabbits using a Percoll gradient method described by Sims27Sims NR Selective impairment of respiration in mitochondria isolated from brain subregions following transient forebrain ischemia in the rat.J Neurochem. 1991; 56: 1836-1844Crossref PubMed Scopus (113) Google Scholar with minor modifications. The isolation buffer contained 225 mmol/L mannitol, 75 mmol/L sucrose, 0.5 mmol/L ethylenediamine tetraacetic acid (EDTA), 5 mmol/L HEPES, and 1 mg/ml fatty acid-free bovine serum albumin (BSA), pH adjusted to 7.3 with KOH. Spinal cord tissue was homogenized using a glass/glass homogenizer in isolation buffer containing 12% Percoll and carefully layered on the top of a 12%/24%/42% discontinuous gradient of Percoll. After 11 minutes of centrifugation at 31,000 × g, the mitochondrial fraction was collected from the top of the 42% Percoll layer of the gradient and then washed twice. For the final wash, we used isolation buffer with BSA omitted and the EDTA concentration reduced to 0.1 mmol/L. All isolation procedures were performed at 0 to 2°C. During experimentation, mitochondria were stored on ice at a final concentration of 15 to 20 mg protein/ml in isolation medium until use. The protein concentration in each preparation was determined by the Biurett method using a plate reader.Neurological AssessmentNeurological status was scored by assessment of hindlimb neurological function at 1 hour after the reperfusion procedure. Animals were classified on a 5-point scale according to the methods of Johnson et al28Johnson SH Kraimer JM Graeber GM Effects of flunarizine on neurological recovery and spinal-cord blood-flow in experimental spinal-cord ischemia in rabbits.Stroke. 1993; 24: 1547-1553Crossref PubMed Scopus (94) Google Scholar: 0, hindlimb paralysis, no voluntary hindlimb movement; 1, severe paraparesis, movement of joints perceptible; 2, functional movement, no hop, active movement but unable to sit without assistance; 3, ataxia, able to sit but disconjugate hop; 4, minimal ataxia and weak hop; and 5, normal neurological function and complete recovery of hindlimb function. The neurofunctional score was evaluated nonparametrically.Transmission Electron MicroscopyFor electron microscopy study, thin sections of ventral horn were cut from Epon tissue blocks, fixed in 2% glutaraldehyde, and subjected to postfixation with 1% osmium tetroxide in 0.1 mol/L cacodylate. Formvar-coated copper grids were stained with 2% uranyl acetate, which was followed by subsequent dehydration with series of ethanol. The samples were embedded overnight with a 1:1 ratio of propylene oxide to epoxy resin. Thereafter, this solution was replaced with 100% epoxy resin and then polymerized in a dry oven at 60°C. Ultrathin sections (70 nm thick) from the embedded samples were imaged by use of a Philips (Bothell, WA) CM 120 transmission electron microscope. Mitochondria number and mitochondria area were determined, and more than 100 mitochondria were examined under each experimental condition. The percentages of mitochondrial membrane damage and mitochondrial swelling were determined by methods described previously.12Kristian T Siesjo BK Calcium-related damage in ischemia.Life Sci. 1996; 59: 357-367Crossref PubMed Scopus (187) Google ScholarChemiluminescent Measurement of Superoxide and Hydrogen Peroxide (H2O2) Production by Intact MitochondriaBis(N-methylacridinium) (lucigenin) and 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) were purchased from Sigma (St. Louis, MO). Lucigenin- or luminol-derived chemiluminescence was monitored with a Berthold Biolumat LB 9505 at 37°C. For measurement of lucigenin-derived chemiluminescence, the reaction mixture contained 0.1 mg of mitochondrial protein in 1 ml of air-saturated respiration buffer in the presence of exogenous substrate (6 mmol/L succinate) with or without the pharmacological uncoupling agent carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (Sigma).29Yang SQ Zhu H Li YB Lin HZ Gabrielson K Trush MA Diehl AM Mitochondrial adaptations to obesity-related oxidant stress.Arch Biochem Biophys. 2000; 378: 259-268Crossref PubMed Scopus (322) Google Scholar The respiration buffer contained 70 mmol/L sucrose, 220 mmol/L mannitol, 2 mmol/L HEPES, 2.5 mmol/L KH2PO4, 2.5 mmol/L MgCl2, 0.5 mmol/L EDTA, and 0.1% BSA, pH 7.4. The lucigenin-derived chemiluminescence was initiated by adding 5 μmol/L lucigenin and was continuously monitored for 40 to 60 minutes. This is a non-redox-cycling concentration of lucigenin.29Yang SQ Zhu H Li YB Lin HZ Gabrielson K Trush MA Diehl AM Mitochondrial adaptations to obesity-related oxidant stress.Arch Biochem Biophys. 2000; 378: 259-268Crossref PubMed Scopus (322) Google Scholar For measurement of luminol-derived chemiluminescence, the reaction mixture contained 0.1 mg of mitochondrial protein in 1 ml of air-saturated respiration buffer plus succinate (6 mmol/L) and horseradish peroxidase (10 μg/ml). The luminol-derived chemiluminescence was initiated by adding 10 μmol/L luminol and was continuously monitored for 40 to 60 minutes.Determination of 8-OxoG Generation8-Oxo-7,8-dihydrodeoxyguanine (8-oxoG) expression in spinal cord tissue was determined as we previously described.30Kloner RA Przyklenk K Whittaker P Deleterious effects of oxygen radicals in ischemia/reperfusion: resolved and unresolved issues.Circulation. 1989; 80: 1115-1127Crossref PubMed Scopus (508) Google Scholar Briefly, the slides were fixed and washed with phosphate-buffered saline (PBS), covered by anti-8-oxoG antibody overnight at 4°C. The slides were stained with diaminobenzamide tetrahydrochloride and counterstained with Methyl Green. The slides were mounted with appropriate mounting media.Western Blot AnalysisWestern blot analysis (wb) was performed as we previously described.26Lin RX Roseborough G Dong YF Williams GM Wei CM DNA damage and repair system in spinal cord ischemia.J Vasc Surg. 2003; 37: 847-858Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 31Wei C Jiang SW Lust JA Daly RC McGregor CGA Genetic expression of endothelial nitric oxide synthase in human atrial myocardium.Mayo Clin Proc. 1996; 71: 346-350Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Tissue samples were homogenized in a lysis buffer (0.1 mol/L NaCl, 0.01 mol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, and 1 μg/ml aprotinin), and then the homogenates were centrifuged at 7000 × g for 15 minutes at 4°C. Supernatants were used as protein samples. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed in a 10% polyacrylamide gel under nonreducing conditions. In brief, protein samples were boiled at 100°C in 2.5% SDS and 5% β-mercaptoethanol, and lysates equivalent to 20 μg of protein from each sample were run on the gel for 90 minutes at 20 mA together with a size marker (rainbow-colored protein; Amersham). The electrophoresis running buffer contained 25 mmol/L Tris base, 250 mmol/L glycine, and 0.1% SDS. Proteins on the gel were transferred to a nitrocellulose membrane with a transfer buffer that consisted of 48 mmol/L Tris base, 39 mmol/L glycine, 0.4% SDS, and 20% methanol. After transfer, membranes were placed in 1% powdered milk in PBS to block nonspecific binding. After reacting with the primary and secondary antibodies, the membrane was subjected to the Enhanced Chemiluminescence analysis system from Amersham. Polyclonal antibody against OGG1 was obtained from Novus Biological (Littleton, CO). Monoclonal antibodies against actin (Ab-6; Oncogene Research Products, MA) were used as controls for equal protein loading. To ascertain specific binding of the antibody for the protein, another membrane was stained without the primary antibody.Immunohistochemical StainingImmunohistochemical staining was performed as we previously described.26Lin RX Roseborough G Dong YF Williams GM Wei CM DNA damage and repair system in spinal cord ischemia.J Vasc Surg. 2003; 37: 847-858Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 31Wei C Jiang SW Lust JA Daly RC McGregor CGA Genetic expression of endothelial nitric oxide synthase in human atrial myocardium.Mayo Clin Proc. 1996; 71: 346-350Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Briefly, after samples were deparaffinized, spinal cord sections were rinsed in 0.1 mol/L PBS for 20 minutes and blocked in 2% normal horse serum for 2 hours. The sections were then incubated with primary antibody OGG1 (1:100; Novus), caspase-3 (1:100; EMD Biosciences, San Diego, CA), AIF (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or polyadenosine-ribose polymerase (PARP-1) (1:100; TM of Trevigen, Gaithersburg, MD) in 10% normal horse serum or 10% normal rabbit serum and 0.3% Triton-X 100 for 20 hours at 4°C. After endogenous peroxidase activity was quenched by exposure of the slides to 0.3% H2O2 and 10% methanol for 20 minutes, the slides were washed in PBS and incubated with secondary antibody-horseradish peroxidase conjugate (Tago, Burlingame, CA). The final reaction was achieved by incubating the sections with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma) dissolved in dimethyl-formamide and sodium acetate. The sections were counterstained with hematoxylin, mounted, and reviewed with an Olympus microscope. Two trained independent observers reviewed these sections. Sample identities were concealed during scoring, and at least three samples were scored per group. The presence of positive immunohistochemical staining was assessed by microscopic examination of the final slides and evaluated by percentage of positive staining in the ventral areas of the spinal cord (0 to 100%). The specificity of positive staining was further confirmed by substitution of normal rabbit serum instead of primary antiserum.Determination of OGG1 ActivityThe activities of DNA repair enzymes in spinal cord tissue were determined by gel-retardation binding assay using A/8-oxoG containing DNA.32Lu AL Repair of A/G and A/8-oxoG mismatches by MutY adenine DNA glycosylase.DNA Repair Protocols. 2000; 152: 3-16Crossref Scopus (23) Google Scholar Oligonucleotide substrates (20 nucleotides) were synthesized and constructed as follows: A, A/G 5′-CCGAGGAATTAGCCTTCTG-3′ and 3′-GCTCCTTAAGCGGAAGACG-5′; B, A/GO 5′-CCGAGGAATTAGCCTTCTG-3′ and 3′-GCTCCTTAAOCGGAAGACG-5′ (O for 8-oxoG); C, C/GO 5′-CCGAGGAATTCGCCTTCTG-3′ and 3′-GCTCCTTAAOCGGAAGACG-5′; and D, C/G 5′-CCGAGGAATTCGCCTTCTG-3′ and 3′-GCTCCTTAAGCGGAAGACG-5′.Duplex A to C contains an A/G, A/8-oxoG, and C/8-oxoG base-base mismatches, respectively, whereas duplex D is a homoduplex containing no mismatch. These oligonucleotides can be uniquely labeled at the 5′ end with polynucleotide kinase and [γ-32P]ATP or labeled at the 3′ end with Klenow fragment and [α-32P]dCTP.OGG1 glycosylase activity was performed according to Hazra et al,33Hazra TK Izumi T Maidt L Floyd RA Mitra S The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation.Nucleic Acids Res. 1998; 26: 5116-5122Crossref PubMed Scopus (162) Google Scholar except that excess DNA substrates containing A/8-oxoG mismatches were added. A 25-μl DNA binding reaction contained 20 μg of protein, 25 mmol/L HEPES (pH 7.6), 50 mmol/L KCl, 2.5 mmol/L EDTA, 2 mmol/L dithiothreitol, and 2.5% glycerol. Two picomoles of unlabeled DNA substrates containing A/8-oxoG mismatches was added before adding 10 fmol of C/8-oxoG-containing DNA labeled at the 8-oxoG strand. The reactions were incubated at 37°C for 1 hour, terminated by phenol-chloroform extraction and ethanol precipitation. Samples were dissolved in 3 μl of sequencing dye. After heating at 90°C for 3 minutes, samples were resolved on a 14% polyacrylamide-8.3 mol/L urea DNA sequencing gel and analyzed by autoradiography.To Determine Apoptosis and Apoptosis PathwaysTo detect DNA fragmentation in situ, nick-end labeling (TUNEL staining) was performed according to our previously reported method30Kloner RA Przyklenk K Whittaker P Deleterious effects of oxygen radicals in ischemia/reperfusion: resolved and unresolved issues.Circulation. 1989; 80: 1115-1127Crossref PubMed Scopus (508) Google Scholar using an ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Briefly, the nuclei of the tissue sections were stripped of proteins by incubating with 20 μg/ml proteinase K for 10 minutes after deparaffinization. After treating with 0.3% H2O2 in distilled water for 5 minutes, the sections were mixed with terminal deoxynucleotidyl transferase buffer (30 mmol/L Tris, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride; Boehringer Mannheim, Indianapolis, IN) containing terminal deoxynucleotidyl transferase enzyme (0.5 U/ml; Boehringer Mannheim) and biotin-16-dUTP (0.04 mmol/L; Boehringer Mannheim) containing 30 mmol/L cobalt chloride and incubated in a humidified chamber at 37°C for 120 minutes. The reaction was terminated by incubating with 300 mmol/L NaCl and 30 mmol/L sodium citrate for 15 minutes at 25°C. After washing with 50 mmol/L Tris-HCl, pH 7.7, sections were developed with diaminobenzadine/H2O2 solution. Counterstaining was performed with hematoxylin. After three washes in Tris-HCl, pH 7.7, the sections were dehydrated in ascending ethanol series. After immersion in xylene, the sections were coverslipped in Permount. To determine the number of motor neurons that underwent apoptosis, the positive or negative motor neurons in the TUNEL staining were counted. The caspase-dependent (caspase-3) and caspase-independent (AIF and PARP) apoptotic pathways were investigated by immunohistochemical staining for appropriate proteins as described above.Statistical AnalysisResults of the quantitative studies are expressed as means ± SEM. Statistical comparisons within each group were performed by using analysis of variance for repeated measures followed by Fisher's least-significant difference test of repeated measures when appropriate. Comparisons between groups were performed by using factorial analysis of variance followed by Fisher's least-significant difference test of repeated measures. We analyzed the neurological score nonparametrically by using an unpaired Student's t-test. Statistical significance was accepted at P < 0.05.ResultsClinical OutcomeTable 1 indicates systolic and diastolic arterial pressures in both groups at baseline, ischemia, and reperfusion interval. There were no significant differences between groups at those intervals. Mean arterial blood pressure was always maintained between 60 and 80 mmHg.Table 1Systolic and Diastolic Arterial Pressures in Both Groups at Baseline, Ischemia, and Reperfusion IntervalSCI group (mmHg)SCI+DZ group (mmHg)SystolicDiastolicSystolicDiastolicBaseline79 ± 546 ± 584 ± 454 ± 3Ischemia82 ± 1060 ± 779 ± 851 ± 9Reperfusion75 ± 352 ± 375 ± 750 ± 8SCI, spinal" @default.
• W2170146710 created "2016-06-24" @default.
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• W2170146710 date "2006-05-01" @default.
• W2170146710 modified "2023-09-30" @default.
• W2170146710 title "The Mitochondrial K-ATP Channel Opener, Diazoxide, Prevents Ischemia-Reperfusion Injury in the Rabbit Spinal Cord" @default.
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