Oxidative sầu mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized khổng lồ be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs to exogenous hydroren peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic khổng lồ developing than khổng lồ mature OLs. Mature OLs were able lớn degrade H2O2 faster than developing OLs, suggesting that higher antioxidant enzyme activity might be the basis for their resistance. Catalase expression và activity were relatively constant during oligodendrocyte maturation, whereas glutathione peroxidase (GPx) was upregulated with a twofold khổng lồ threefold increase in its expression và activity. Thus, it appeared that the developmental change in resistance khổng lồ H2O2 was caused by modulation of GPx but not by catalase expression. To thử nghiệm the relative roles of catalase and GPx in the setting of oxidative sầu áp lực, we measured enzyme activity in cells exposed to lớn H2O2 & found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led to an increase in the vulnerability of mature OLs lớn H2O2 as well as a reduction in catalase activity. Finally, H2O2-dependent inactivation of catalase in developing OLs was prevented by the GPx mimic ebselen. These data provide evidence for a key role for GPx-catalase cooperativity in the resistance of mature OLs to lớn H2O2-induced cell death.
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Ischemia/reperfusion injury lớn the developing brain is a major cause of neurological disorders in the perinatal period. Neurological deficits in infants surviving hypoxic-ischemic insult include mental retardation, seizures, & cerebral palsy. The neuropathology of perinatal brain injury is complex, & involves gray và Trắng matter to varying degrees, depending on the gestational age và the developmental stage of cerebral vascularity (Kinney and Armsvào, 1997). Periventricular leukomalacia (PVL) involves injury khổng lồ the cerebral trắng matter resulting in a chronic disturbance of myelination; it is the predominant underlying pathology of cerebral palsy in premature infants (Volpe, 1997, 2001). During the maturational period of peak vulnerability lớn PVL, the Trắng matter is predominantly populated with premyelinating oligodendrocytes (OLs) (Baông chồng et al., 2002).
In human PVL, there is now emerging evidence that oxidative sầu bao tay plays a key role in the pathogenesis of the white-matter injury (Haynes et al., 2003). Previous work has shown that developing OLs are more vulnerable than mature OLs lớn endogenous oxidative stress caused by the depletion of intracellular glutathione (GSH). (Oka et al., 1993; Yonezawa et al., 1996; Baông chồng et al., 1998, 2001). However, the sensitivity of OLs at specific stages of development khổng lồ exogenous sources of oxidative bao tay remains unknown.
Peroxides, including hydrogen peroxide (H2O2), are one of the main reactive oxygen species (ROS) leading to lớn oxidative sầu stress (Halliwell and Gutteridge, 1999). H2O2 is continuously generated by several enzymes (including superoxide dismutase, glucose oxidase, and monoamine oxidase) & must be degraded lớn prsự kiện oxidative damage. The cytotoxic effect of H2O2 is thought to lớn be caused by hydroxyl radicals generated from iron-catalyzed reactions, causing subsequent damage khổng lồ DNA, proteins, và membrane lipids (Halliwell, 1992). Recently, both glutathione peroxidase và catalase, the two main enzymes involved in H2O2 detoxification, have been shown khổng lồ be implicated in the disposal of exogenous H2O2 by astrocytes (Dringene và Hamprecht, 1997). Both have sầu been found in the brain (De Marchemãng cầu et al., 1974; Gaunt and de Duve sầu, 1976; Brannan et al., 1981) & in astrocytes & oligodendrocytes (Drinren & Hamprecht, 1997; Hirrlinger et al., 2002). Catalase is known khổng lồ be of special importance when the clearance of H2O2 in high concentrations is required. More recently, a higher capathành phố for the degradation of H2O2 has been demonstrated in myelin basic protein (MBP) expressing oligodendrocytes than in astrocytes, microglia, or neurons (Hirrlinger et al., 2002). However, the developmental variation in the vulnerability of OLs lớn H2O2 has not been investigated.
In the present work, we investigated the sensitivity of OLs khổng lồ H2O2 at specific developmental stages of maturation. Developing OLs appeared to lớn be more vulnerable lớn H2O2 than were mature OLs. In pursuing the basis for this difference in vulnerability, we found that GPx expression was upregulated in mature OLs. Although catalase expression was unchanged through development, this enzyme in developing OLs was inactivated by exposure lớn H2O2. The vulnerability of catalase lớn H2O2 appeared to lớn depkết thúc on the cấp độ of GPx activity. In mature OLs, the upregulation of glutathione peroxidase maintains the catalase activity at a high màn chơi in the presence of H2O2.
Materials and Methods
Materials. DMEM, HBSS, Earle"s balanced salt solution (EBSS), fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen (Rockville, MD). Unless otherwise specified, all other chemicals were from Sigma (St. Louis, MO).
Oligodendrocyte cultures. Primary rat OLs were prepared from the cerebral hemispheres of Sprague Dawley rats at postnatal day 1-2 using a shaking method (McCarthy & de Vellis, 1980; Oka et al., 1993) with modifications, as described previously (Li et al., 2003). Briefly, forebrains free of meninges were chopped inlớn 1 mm3 blocks & placed into HBSS containing 0.01% trypsin and 10 μg/ml DNase. After digestion for 15 min at 37°C, the tissue was collected by centrifugation và triturated in the plating medium (DMEM 20% serum) containing DMEM, 20% fetal bovine serum, 40 IU/ml penicillin, & 40 μg/ml streptomycin, và passed through a 70 μm sieve sầu. Cells were plated onlớn polylysine-coated 75 cm2 flasks at a density of 1 pup brain per flask. Cultures were fed with fresh DMEM 20S medium every other day for 10-11 d at 37°C in a humid atmosphere of 5% CO2 & 95% air.
To isolate OLs, the mixed glial cultures were shaken for 1 hr at 200 rpm at 37°C to lớn remove adherent microglia/macrophages, and the cultures were washed with the same medium and subjected khổng lồ shaking at 200 rpm overnight (18-22 hr) khổng lồ separate OLs from the astrocyte layer. The suspension was plated onlớn uncoated Petri dishes and incubated for 1 hr at 37°C lớn further remove residual microglia & astrocytes that adhere to lớn the dishes. The OLs were then collected by passing through a 15 μm sieve sầu, followed by centrifugation. Isolated OLs were plated onto lớn poly-d-ornithine (50 μg/ml)-coated 96 well culture plates (at density of 3.3 × 103 cells per well for cell-survival assay), 24 well plates with glass coverslips (1.74 × 104 cells per well for imaging), and 60 mm plates (2.75 × 105 cells per plate for enzyme assays). Purified OLs were cultured for 7-8 d in a serum-không lấy phí basal defined medium (BDM): DMEM, 0.1% bovine serum albumin, 50 μg/ml apo-transferrin, 50 μg/ml insulin, 30 nm sodium selenite, 10 nm d-biotin, 10 nm hydrocortisone, 200 μm l-cystine, 10 ng/ml platelet-derived growth factor, and 10 ng/ml basic fibroblast growth factor. At 7-8 d, the cultures were composed primarily of progenitors and pre-OLs (A2B5+, O4+, O1-, MBP-negative). After 7 d, culture medium was changed lớn serum-không tính phí BDM containing 10 ng/ml ciliary neurotrophic factor and 15 nm 3,3′,5-triiodo-l-thyronine for 14 additional days until cells were differentiated inkhổng lồ mature OLs (MBP+). The purity of OL cultures was consistently >95% OLs with 2O2 diluted from a 9.8 m stoông xã solution. Unless otherwise specified, the cells were incubated for 24 hr before being assayed for cell survival. Alternatively, H2O2 was generated in OL cultures by the addition of glucose oxidase (đôi mươi mU/ml) khổng lồ the medium (BDM; 25 mm glucose) (Klặng and Kyên ổn, 1991). The cells were incubated for 24 hr followed by Alamar xanh assay (Southern Biogiải pháp công nghệ, Birmingđê mê, AL) for cell viability.
Cell-survival assay. Cell survival was determined after treatment for 24 hr using Alamar blue, a tetrazolium dye that is reduced by living cells to lớn a fluorescent product. This assay is similar in principle to the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay và has been previously validated as an accurate measure of survival of OLs (Back et al., 1999). For the H2O2 challenge, the results from the Alamar xanh assay were matched by a calcein-based assay (Molecular Probes, Euren, OR) (data not shown). All results of cell death assays were confirmed by visual inspection using phase-contrast microscopy. Briefly, culture medium in the 96 well plate was aspirated, và cells were incubated with 200 μl of assay solution prepared by diluting 100× stoông xã solution of Alamar xanh inlớn EBSS for 2 hr at 37°C. The fluorescence of the assay solution, reflecting cell viability, was measured with a fluorescence plate reader (FluoroCount, Packard Instrument, Meridan, CT) using an excitation wavelength of 560 nm và an emission wavelength of 590 nm. All survival assays were performed at least in triplicate.
Intracellular free radical accumulation measurement. Intracellular không tính phí radical generation was evaluated with dichlorohydrofluorescein diacetate (DCFA) (C-2938; Molecular Probes) (Wang và Joseph, 1999). DCFA was prepared as a 100 mm stoông xã in dimethylsulfoxide, and stored in the dark at -20°C. The cells were loaded with 100 μl of a 100 μm DCFA solution & incubated for 20 min at 37°C. Next, the cells were washed again in EBSS lớn remove all excess DCFA that had not penetrated the cells. The baseline fluorescence was measured at the excitation & emission wavelengths of 485 & 530 nm, respectively, using a multiwell fluorescence plate reader. The fluorescence response of the cells was compared across stages at various concentrations of H2O2 by calculating the percent increase in fluorescence, comparing baseline with 1 hr of exposure.
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H2O2 colorimetric assay. A method described previously was used lớn characterize the kinetics of H2O2 depletion from the culture medium. The OLs were plated in polyornithine-coated 24 well plates. H2O2 depletion was monitored by a colorimetric assay that involved the oxidation of o-dianisidine dihydrochloride (o-DD) in the presence of horseradish peroxidase (Yakes & Van Houten, 1997). At the start of the experiment, H2O2 was added khổng lồ each well containing 500 μl EBSS lớn yield 400 μm. In this medium, the hydrogen peroxide concentration remained constant over the period studied in wells lacking any cells. As the experiment progressed, 100 μl aliquots of the treatment medium were removed at specific time points. These aliquots were then incubated with 2.5 U/ml of horseradish peroxidase và 250 μm o-DD for 1 hr at 37°C. The absorbance was then measured at 470 nm against a blank containing EBSS without the addition of H2O2. The amount of H2O2 remaining in the medium was determined from a standard curve generated by using known concentrations of H2O2. The initial rate of H2O2 clearance was obtained manually from the plotted results.
Immunocytochemistry, Tdt-mediated dUTPhường nichồng over labeling, và immunofluorescence microscopy. Cells were washed in cold PBS và fixed with 4% paraformaldehyde for 10 min at room temperature, washed three times with PBS, & blocked with PBS containing 5% goat serum for 1 hr at room temperature. The coverslips were incubated for 1 hr with mouse monoclonal antibodies O4 and O1 (1:100 dilution; gifts of Dr. Steven E. Pfeiffer, University of Connecticut Health Center, Farmington, CT), MBP (1:500 dilution; Boehringer Mannheyên ổn, Indianapolis, IN). After three to four washes at 15 min each, the appropriate secondary antitoàn thân conjugated with Alexa red (1:1000 dilution; Molecular Probes) was added lớn the coverslips & incubated for 1 hr. After extensive washes, cells were again exposed briefly khổng lồ 4% paraformaldehyde in PBS for 5 min at room temperature. After several rinses in PBS, the coverslips were then incubated for 2 hr with PBS containing 5% goat serum & 0.1% Triton X-100 containing rabbit polyclonal anti-catalase (1:500 dilution; Abcam, Cambridge, UK) antibodies. After three washes, the appropriate secondary antitoàn thân conjugated with Oregon green (1:500 dilution; Molecular Probes) was incubated with cells for 1 hr. Nuclei were finally stained by adding Hoechst 33258 at a final concentration of 2 μg/ml for 1 min. After three more washes, the coverslips were mounted onto lớn glass slides with FluoroMount (Southern Biotechnology, Birmingmê mệt, AL) and kept in the dark at 4°C.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP.. niông chồng over labeling (TUNEL) staining was performed using the In Situ Cell Death detection kit from Roche (Indianapolis, IN) và following the protocol for cell culture.
Cell images were captured with a fluorescence microscope (Eclipse E800; Nikon, Tokyo, Japan) equipped with a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Protein extracts & Western blot. OLs were rinsed with cold PBS and scraped into lysis buffer containing the following: 150 milimet NaCl, 50 mm Tris, 0.5% Triton X-100, & 1× protease inhibitor mixture (Roche). For Western blotting, samples were boiled for 5 min in the presence of β-mercaptoethanol and SDS; 10 μg of protein from the cell lysates were separated by electrophoresis using a 12% polyacrylamide gel. After electroblotting khổng lồ a polyvinylidene difluoride membrane and blocking of nonspecific binding sites, membranes were exposed either lớn anti-catalase (1:500 dilution; Abcam, Cambridge, UK) or khổng lồ anti-GPx-1 (1:100 dilution; Novus Biological, Littleton, CO) antibodies followed by appropriate secondary antibodies conjugated with horseradish peroxidase and detection by chemiluminescence (PerkinElmer, Wellesley, MA).
Catalase, GPx, và glutathione reductase enzyme activity assays; glutathione chemical assay. Briefly, cells were harvested & in a lysate buffer containing 0.1% Triton X-100, then centrifuged to lớn remove sầu the supernatant for assay. Catalase activity measurement was based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced was measured spectrophotometrically at 540 nm. One unit of catalase was defined as the formation of 1 nmol of formaldehyde per minute per milligram of protein. GPx activity was measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione produced on reduction of hydroperoxide by GPx was recycled khổng lồ its reduced size by the oxidation of NADPH to lớn NADP+, which was accompanied by a decrease in absorbance at 340 nm. Under conditions of the assay, the rate of decrease in the absorbance at 340 nm was directly proportional to lớn the GPx activity in the sample. One unit of GPx was defined as the amount of enzyme causing the oxidation of 1 nmol of NADPH per minute và per milligram of protein. Catalase and GPx activities were measured using kits from Cayman Chemical (Ann Harbor, MI). Glutathione reductase activity was assayed in the presence of 1 milimet oxidized GSH (GSSG) by measuring the rate of NADPH oxidation, which is accompanied by a rapid decrease in absorbance at 340 nm. The assay temperature is 25°C. Results were expressed similarly khổng lồ those of the GPx assay.
Total intracellular reduced GSH was measured by a fluorometric method as described previously (Wang & Joseph, 1999).
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Data collected was analyzed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). Unless otherwise indicated, the results shown are from one experiment representative sầu of three khổng lồ six experiments. Either Student"s t demo, one-way ANOVA, or two-way ANOVA with the Bonferroni post-demo comparison were performed for statistical analyses.
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