Journal of Neuroscience 18 February 2004, 24 (7) 1531-1540; DOI: https://doi.org/10.1523/infobandarpkr.comle.3989-03.2004
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Abstract

Oxidative sầu mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized lớn be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs to lớn exogenous hydrogene peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic to developing than to mature OLs. Mature OLs were able khổng lồ 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 to threefold increase in its expression and 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 chạy thử the relative roles of catalase & GPx in the setting of oxidative sầu bao tay, we measured enzyme activity in cells exposed lớn H2O2 & found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led khổng lồ 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 lớn H2O2-induced cell death.Quý khách hàng sẽ xem: Enzyme peroxidase là gì

Introduction

Ischemia/reperfusion injury khổng lồ 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, and cerebral palsy. The neuropathology of perinatal brain injury is complex, and involves gray and white matter to varying degrees, depending on the gestational age và the developmental stage of cerebral vascularity (Kinney and Armstrong, 1997). Periventricular leukomalacia (PVL) involves injury lớn 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 khổng lồ PVL, the Trắng matter is predominantly populated with premyelinating oligodendrocytes (OLs) (Back et al., 2002).

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In human PVL, there is now emerging evidence that oxidative găng 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 bức xúc 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 to exogenous sources of oxidative sầu bao tay remains unknown.

Peroxides, including hydrogene peroxide (H2O2), are one of the main reactive sầu oxygene species (ROS) leading to oxidative căng thẳng (Halliwell và Gutteridge, 1999). H2O2 is continuously generated by several enzymes (including superoxide dismutase, glucose oxidase, và monoamine oxidase) và must be degraded to lớn prevent oxidative sầu damage. The cytotoxic effect of H2O2 is thought khổng lồ be caused by hydroxyl radicals generated from iron-catalyzed reactions, causing subsequent damage lớn DNA, proteins, và membrane lipids (Halliwell, 1992). Recently, both glutathione peroxidase và catalase, the two main enzymes involved in H2O2 detoxification, have sầu been shown khổng lồ be implicated in the disposal of exogenous H2O2 by astrocytes (Drinren và Hamprecht, 1997). Both have been found in the brain (De Marchena et al., 1974; Gaunt và de Duve sầu, 1976; Brannan et al., 1981) và in astrocytes và oligodendrocytes (Drinren and Hamprecht, 1997; Hirrlinger et al., 2002). Catalase is known to be of special importance when the clearance of H2O2 in high concentrations is required. More recently, a higher capacity 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 khổng lồ H2O2 has not been investigated.

In the present work, we investigated the sensitivity of OLs lớn H2O2 at specific developmental stages of maturation. Developing OLs appeared lớn be more vulnerable khổng lồ 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 to lớn H2O2. The vulnerability of catalase to lớn H2O2 appeared khổng lồ depkết thúc on the màn chơi 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, và 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 và de Vellis, 1980; Oka et al., 1993) with modifications, as described previously (Li et al., 2003). Briefly, forebrains không tính phí of meninges were chopped into lớn 1 mm3 blocks and placed inlớn HBSS containing 0.01% trypsin and 10 μg/ml DNase. After digestion for 15 min at 37°C, the tissue was collected by centrifugation & triturated in the plating medium (DMEM 20% serum) containing DMEM, 20% fetal bovine serum, 40 IU/ml penicillin, and 40 μg/ml streptomycin, và passed through a 70 μm sieve. Cells were plated onto lớn polylysine-coated 75 cmét vuông 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 to lớn shaking at 200 rpm overnight (18-22 hr) to lớn separate OLs from the astrocyte layer. The suspension was plated onlớn uncoated Petri dishes & incubated for 1 hr at 37°C lớn further remove sầu 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 onlớn 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 milimet plates (2.75 × 105 cells per plate for enzyme assays). Purified OLs were cultured for 7-8 d in a serum-miễn 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, và 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 khổng lồ serum-free BDM containing 10 ng/ml ciliary neurotrophic factor & 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 stock 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 (trăng tròn mU/ml) khổng lồ the medium (BDM; 25 mm glucose) (Kim and Kyên ổn, 1991). The cells were incubated for 24 hr followed by Alamar blue assay (Southern Biocông nghệ, Birmingham mê, AL) for cell viability.

Cell-survival assay. Cell survival was determined after treatment for 24 hr using Alamar xanh, a tetrazolium dye that is reduced by living cells khổng lồ a fluorescent hàng hóa. This assay is similar in principle lớn the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay and has been previously validated as an accurate measure of survival of OLs (Bachồng 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 chồng solution of Alamar blue 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 and an emission wavelength of 590 nm. All survival assays were performed at least in triplicate.

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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 to each well containing 500 μl EBSS khổng lồ yield 400 μm. In this medium, the hydroren 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 and 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 sầu generated by using known concentrations of H2O2. The initial rate of H2O2 clearance was obtained manually from the plotted results.

Immunocytochemistry, Tdt-mediated dUTPhường. niông xã kết thúc labeling, and immunofluorescence microscopy. Cells were washed in cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, washed three times with PBS, and 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 & O1 (1:100 dilution; gifts of Dr. Steven E. Pfeiffer, University of Connecticut Health Center, Farmington, CT), MBP (1:500 dilution; Boehringer Mannheyên, Indianapolis, IN). After three lớn four washes at 15 min each, the appropriate secondary antibody conjugated with Alexa red (1:1000 dilution; Molecular Probes) was added khổng lồ the coverslips and incubated for 1 hr. After extensive sầu washes, cells were again exposed briefly to lớn 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 và 0.1% Triton X-100 containing rabbit polyclonal anti-catalase (1:500 dilution; Abcam, Cambridge, UK) antibodies. After three washes, the appropriate secondary antibody toàn 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 Biogiải pháp công nghệ, Birmingsi, AL) & kept in the dark at 4°C.

Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining was performed using the In Situ Cell Death detection kit from Robít (Indianapolis, IN) & 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 và Western blot. OLs were rinsed with cold PBS & scraped inlớn lysis buffer containing the following: 150 mm NaCl, 50 mm Tris, 0.5% Triton X-100, and 1× protease inhibitor mixture (Roche). For Western blotting, samples were boiled for 5 min in the presence of β-mercaptoethanol & SDS; 10 μg of protein from the cell lysates were separated by electrophoresis using a 12% polyacrylamide gel. After electroblotting lớn a polyvinylidene difluoride membrane and blocking of nonspecific binding sites, membranes were exposed either khổng lồ 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, and glutathione reductase enzyme activity assays; glutathione chemical assay. Briefly, cells were harvested và in a lysate buffer containing 0.1% Triton X-100, then centrifuged to remove 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 to its reduced khung by the oxidation of NADPH to 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 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 & per milligram of protein. Catalase và 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 to those of the GPx assay.

Total intracellular reduced GSH was measured by a fluorometric method as described previously (Wang and Joseph, 1999).