Design

A controlled, cytobiological experiment.

Time and setting

The experiment was performed at Central Laboratory in Shanghai Tongji Hospital, Tongji University School of Medicine, China from January 2011 to January 2012.

Materials

A total of 20 clean, healthy, newborn Sprague-Dawley rats were purchased from the Chinese Academy of Medical Sciences, China (license No. SCXK (Hu) 2008-0016). All experimental protocols were in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[28].

Methods

Rat primary oligodendrocyte lineage cell culture

Purified OPCs were isolated using the shake-off method as previously described with some modifications^[29]. Briefly, newborn (postnatal day 1) rats were decapitated and mixed cortical glial cell cultures were generated from cerebra and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (D10) for 10 days in 75 cm² flasks at 37°C and 5% CO₂ with a medium change every 3 days. Microglia were roughly separated by shaking the culture flasks for 1 hour at  250 r/min on a gyratory shaker at 37°C. Cells were rinsed three times with PBS to remove the detached microglia. The culture flasks were further shaken for an additional 16 hours at 280–290 r/min at 37°C, to separate the OPCs. To remove contaminating microglia and astrocytes in the isolated OPC cultures, the detached cell suspension was seeded and left to adhere twice in uncoated Petri dishes (Sterilin, Staffordshire, UK) for    2 hours at 37°C with 5% CO₂. The collected OPCs in D10 medium were then re-plated at densities between  50 000 and 5 000 cells/cm² onto poly-L-lysine- coated 24-well plates, dishes, or coverslips and maintained at 37°C with 5% CO₂. Finally, to remove any remaining microglia, 6 hours after plating OPC cultures were treated with 5 mM leucine methylester (Sigma, St. Louis, MO, USA) for 15 minutes at room temperature and were then washed three times with DMEM^[30]. Cells were expanded in B104 conditioned medium to further enrich and expand the oligodendrocyte cultures^[14]. For differentiation assays, 30 ng/mL T3 and 40 ng/mL T4 were added to the medium^[31]. As the cultures in differentiation medium contained heterogeneous cells that were in different stages including OPCs and mature oligodendrocytes, we adopted the term “oligodendrocyte lineage cells” to define the whole population in the cultures^[32].

Immunofluorescence staining of myelin basic protein, NG2, A2B5, TfR and F-H expression in oligodendrocytes

Immunocytochemistry of iron-related proteins and oligodendrocyte lineage cells was performed as described previously^{[15, 33-34]}. After 3 days of culture in differentiation medium, oligodendrocytes were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with 0.3% Triton X-100 in PBS for 5 minutes. Cells were incubated with mouse anti-rat myelin basic protein monoclonal antibodies (1:100; Millipore, Billerica, MA, USA), rabbit anti-NG2 polyclonal antibody (1:200; Chemicon, Temecula, CA, USA) and rabbit anti-A2B5 polyclonal antibody (1:100; Chemicon) overnight at 4°C. Final detection was performed by incubation with FITC conjugated goat anti-rabbit IgG antibodies (Sigma) or TRITC-conjugated goat anti-mouse IgG antibodies diluted 1:400 in PBS containing 5% normal goat serum for 1 hour at 37°C. Cells were counted after Hoechst33342 nuclear staining (Sigma). An Olympus microscope (IX70) connected to a computer equipped with a charge-coupled device camera (Olympus Corporation, Japan) was used to visualize and capture images, which were then analyzed by image analysis software (MetaMorph version 4, Universal Imaging Corporation, Auburn, WA, USA). For NG2⁺ or myelin basic protein⁺ cell number analysis, cells from 10 randomly captured fields were analyzed per well of triplicate wells for individual treatments in three independent experiments, counting approximately 3 000 cells for each data point. Mean numbers of NG2⁺ or BMP⁺ cells per field of view were normalized to controls and the results were expressed as relative to controls.

Western blot analysis of myelin basic protein, TfR and F-H expression in oligodendrocytes

For myelin basic protein^[32], TfR^[35] and F-H ^{[26, 36]} expression analysis, OPCs were allowed to differentiate for 3 days in differentiation medium. Whole-cell lysates were obtained by radioimmune precipitation assay with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM phenylmethyl sulfonylfluoride, 1 × Roche complete mini protease inhibitor) on ice. Lysates were centrifuged at 12 000 × g for 10 minutes at 4°C, and protein concentration in the supernatant was determined by bicinchoninic acid assay (Sigma). After boiling for 5 minutes, 15 μg of total protein from whole-cell lysates was separated by 15% SDS-PAGE denaturing gel and were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Inc. Dassel, Germany). Membranes were blocked with 10% nonfat dried milk in Tris-buffered saline containing Tween-20 (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.4) for 1 hour, and immunoblotted with mouse anti-myelin basic protein monoclonal antibodies (Millipore, Billerica, MA, USA), mouse anti-β-actin monoclonal antibodies (Abcam, Cambridge, MA, USA), rabbit anti-TfR polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit anti-F-H polyclonal antibodies (Santa Cruz Biotechnology) at a dilution of 1:1 000 overnight at 4°C. Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG antibodies (1:400; Santa Cruz Biotechnology) were used for immunoblotting to allow the detection of the final signal with enhanced chemiluminescence reagents (Pierce Biotechnology, Rockford, IL, USA). Quantifications of myelin basic protein, TfR and F-H expression were performed by scanning densitometric analysis on myelin basic protein, TfR and F-H relative to actin levels. Scanning absorbances were normalized and expressed as relative fold to controls. Results were from three independent experiments.

Statistical analysis

Data are expressed as mean ± SEM from at least three independent experiments unless otherwise indicated. Differences between groups were statistically tested by one-way analysis of variance followed by two-tailed t-test. A value of P < 0.05 was considered statistically significant.

(1) Transferrin receptor and ferritin-H are expressed in oligodendrocyte lineage cells.
(2) Transferrin receptor and ferritin-H are upregulated during maturation of oligodendrocyte lineage cells.
(3) Additional iron does not affect oligodendrocyte precursor cell maturation or ferritin-H expression but decreases the expression of transferrin receptor.

1.少突胶质细胞表达转铁蛋白受体和铁蛋白重链。
2.在少突胶质细胞成熟过程中，转铁蛋白受体和铁蛋白重链表达增加。
3.额外的铁不影响少突胶质前体细胞的成熟，不影响少突胶质前体细胞中铁蛋白重链的表达，但降低了转铁蛋白受体的表达。

研究背景：
脑缺血产生大量氧自由基造成少突胶质细胞损伤。与星形胶质细胞和神经元相比，少突胶质细胞中含有丰富的铁和铁蛋白，而还原型谷胱甘肽含量和谷胱甘肽过氧化物酶活性低。一方面少突胶质细胞中的铁蛋白和转铁蛋白可作为铁螯合剂和抗氧化物保护细胞，另一方面，少突胶质细胞缺血缺氧，促使铁蛋白和转铁蛋白释放铁，通过fenton反应和Haber-Weiss反应产生大量氧自由基，引发脂质过氧化。还原型谷胱甘肽在氧自由基清除中起关键性作用。过氧化物由谷胱甘肽过氧化物酶清除。由于少突胶质细胞铁含量高，清除自由基能力弱而且显著依赖于氧化磷酸化，少突胶质细胞更容易受到氧化应激损伤。由此引发的链式脂质过氧化反应损伤细胞膜和线粒体膜，而产生的脂质过氧化物和细胞毒性副产物等，对轴突和少突胶质细胞也具有毒性。Hollensworth等认为氧化应激还可能损伤线粒体DNA，从而增加细胞色素c释放，激活caspase-9，启动凋亡反应。