Oligodendrocytes and Progenitors Become Progressively Depleted within Chronically Demyelinated Lesions
2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63726-1
ISSN1525-2191
AutoresJeffrey L. Mason, Arrel D. Toews, Janell Hostettler, Pierre Morell, Kinuko Suzuki, James E. Goldman, Glenn K. Matsushima,
Tópico(s)Glioma Diagnosis and Treatment
ResumoTo understand mechanisms that may underlie the progression of a demyelinated lesion to a chronic state, we have used the cuprizone model of chronic demyelination. In this study, we investigated the fate of oligodendrocytes during the progression of a demyelinating lesion to a chronic state and determined whether transplanted adult oligodendrocyte progenitors could remyelinate the chronically demyelinated axons. Although there is rapid regeneration of the oligodendrocyte population following an acute lesion, most of these newly regenerated cells undergo apoptosis if mice remain on a cuprizone diet. Furthermore, the oligodendrocyte progenitors also become progressively depleted within the lesion, which appears to contribute to the chronic demyelination. Interestingly, even if the mice are returned to a normal diet following 12 weeks of exposure to cuprizone, remyelination and oligodendrocyte regeneration does not occur. However, if adult O4+ progenitors are transplanted into the chronically demyelinated lesion of mice treated with cuprizone for 12 weeks, mature oligodendrocyte regeneration and remyelination occurs after the mice are returned to a normal diet. Thus, the formation of chronically demyelinated lesions induced by cuprizone appears to be the result of oligodendrocyte depletion within the lesion and not due to the inability of the chronically demyelinated axons to be remyelinated. To understand mechanisms that may underlie the progression of a demyelinated lesion to a chronic state, we have used the cuprizone model of chronic demyelination. In this study, we investigated the fate of oligodendrocytes during the progression of a demyelinating lesion to a chronic state and determined whether transplanted adult oligodendrocyte progenitors could remyelinate the chronically demyelinated axons. Although there is rapid regeneration of the oligodendrocyte population following an acute lesion, most of these newly regenerated cells undergo apoptosis if mice remain on a cuprizone diet. Furthermore, the oligodendrocyte progenitors also become progressively depleted within the lesion, which appears to contribute to the chronic demyelination. Interestingly, even if the mice are returned to a normal diet following 12 weeks of exposure to cuprizone, remyelination and oligodendrocyte regeneration does not occur. However, if adult O4+ progenitors are transplanted into the chronically demyelinated lesion of mice treated with cuprizone for 12 weeks, mature oligodendrocyte regeneration and remyelination occurs after the mice are returned to a normal diet. Thus, the formation of chronically demyelinated lesions induced by cuprizone appears to be the result of oligodendrocyte depletion within the lesion and not due to the inability of the chronically demyelinated axons to be remyelinated. Although most acutely demyelinated lesions in multiple sclerosis (MS) are remyelinated,1Prineas JW Connell F Remyelination in multiple sclerosis.Ann Neurol. 1979; 5: 22-31Crossref PubMed Scopus (382) Google Scholar the lesions eventually progress to a state of chronic demyelination, characterized by sparse remyelination, few oligodendrocytes, and axon degeneration.2Lucchinetti C Brück W Parisi J Scheithauer B Rodriguez M Lassmann H A quantitative analysis of oligodendrocytes in multiple sclerosis lesions.Brain. 1999; 122: 2279-2295Crossref PubMed Scopus (401) Google Scholar, 3Raine CS Cross AH Axonal dystrophy as a consequence of long-term demyelination.Lab Invest. 1989; 60: 714-725PubMed Google Scholar, 4Ludwin SK Central nervous system remyelination: studies in chronically damaged tissue.Ann Neurol. 1994; 6: S143-S145Crossref Scopus (27) Google Scholar, 5Suzuki K Andrews JM Waltz JM Terry RD Ultrastructure studies of multiple sclerosis.Lab Invest. 1969; 20: 444-454PubMed Google Scholar Although the central nervous system (CNS) has the ability to regenerate new oligodendrocytes that remyelinate demyelinated axons following acute demyelination,6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar, 7Gensert JM Goldman JE Endogenous progenitors remyelinate demyelinated axons in the adult CNS.Neuron. 1997; 19: 197-203Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 8Ludwin SK Central nervous system demyelination and remyelination in the mouse.Lab Invest. 1978; 39: 597-612PubMed Google Scholar, 9Blakemore WF Remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cuprizone.J Neurol Sci. 1973; 20: 73-83Abstract Full Text PDF PubMed Scopus (136) Google Scholar it has been suggested that mature oligodendrocytes are not regenerated within chronically demyelinated lesions due to: 1) the depletion of the oligodendrocyte progenitors;4Ludwin SK Central nervous system remyelination: studies in chronically damaged tissue.Ann Neurol. 1994; 6: S143-S145Crossref Scopus (27) Google Scholar 2) the inability of the progenitors to proliferate and differentiate within the lesion due to aging10Sim FJ Zhao C Penderis J Franklin RJM The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation.J Neurosci. 2002; 22: 2451-2459PubMed Google Scholar or a non-conducive environment;11John GR Shankar SL Shaft-Zagardo B Massimi A Lee SC Raine CS Brosnan CF Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.Nat Med. 2002; 8: 1115-1121Crossref PubMed Scopus (412) Google Scholar, 12Chang A Nishiyama A Peterson J Prineas J Trapp BD NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions.J Neurosci. 2000; 20: 6404-6412Crossref PubMed Google Scholar, 13Scolding N Franklin R Stevens S Heldin C-H Compston A Newcombe J Oligodendrocyte progenitors are present in the normal adult human CNS and in the lesions of multiple sclerosis.Brain. 1998; 121: 2221-2228Crossref PubMed Scopus (263) Google Scholar, 14Wolswijk G Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells.J Neurosci. 1998; 18: 601-609PubMed Google Scholar and/or 3) axon damage3Raine CS Cross AH Axonal dystrophy as a consequence of long-term demyelination.Lab Invest. 1989; 60: 714-725PubMed Google Scholar, 15Bjartmar C Trapp BD Axonal and neuronal degeneration in multiple sclerosis: mechanisms and functional consequences.Curr Opin Neurol. 2001; 14: 271-278Crossref PubMed Scopus (399) Google Scholar, 16Silber E Sharief MK Axonal degeneration in the pathogenesis of multiple sclerosis.J Neurol Sci. 1999; 170: 11-18Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar or the inability of chronically demyelinated axons to be remyelinated.17Chang A Wallace MD Tourtellotte W Rudick R Trapp B Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis.N Engl J Med. 2002; 346: 165-173Crossref PubMed Scopus (816) Google Scholar To test these hypotheses, we used the cuprizone model of chronic demyelination.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar Previously, we reported acute demyelination in C57BL/6 mice18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar, 19Hiremath MM Saito Y Knapp GW Ting JP-Y Suzuki K Matsushima GK Microglia/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice.J Neuroimmunol. 1998; 92: 38-49Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar and the apoptotic death of mature oligodendrocytes within the corpus callosum in mice exposed to cuprizone for a short duration.6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar Once cuprizone is removed from the diet, the mature oligodendrocytes begin to repopulate the lesion and remyelinate the demyelinated axons. This regeneration of the mature oligodendrocyte population is presumably derived from the differentiation of accumulating progenitors within the demyelinating lesion.6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar However, if mice are maintained on the cuprizone diet for a prolonged period of time, remyelination eventually fails.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar In addition, we report here that the newly regenerated mature oligodendrocytes, along with the resident progenitors, become progressively depleted within the chronically demyelinated corpus callosum. However, adult O4+ oligodendrocyte progenitors transplanted into the chronic lesions differentiate into mature oligodendrocytes and remyelinate a large number of the demyelinated axons if the mice are returned to a normal diet following 12 weeks of cuprizone intoxication. Thus, the formation of chronically demyelinated lesions induced by long-term cuprizone toxicity is the result of oligodendrocyte depletion within the lesion and not due to a non-conducive environment or the inability of the chronically demyelinated axons to be remyelinated. Adult male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). At 8 weeks of age, the mice were fed a diet of milled Purina mouse chow containing 0.2% cuprizone (Sigma Chemical Co., St. Louis, MO) by weight for up to 16 weeks to induce chronic demyelination.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar Additional mice were fed cuprizone for 6 weeks and then returned to a normal diet to allow remyelination.6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar, 19Hiremath MM Saito Y Knapp GW Ting JP-Y Suzuki K Matsushima GK Microglia/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice.J Neuroimmunol. 1998; 92: 38-49Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar Some mice were anesthetized and perfused through the heart with 4% paraformaldehyde (PFA). The brains were removed and cut mid-sagittally. Half of the brain was then fixed overnight in 4% PFA at 4°C and then embedded in paraffin. 5-μm sections were cut with a microtome at the fornix region of the corpus callosum, mounted onto gelatin-coated slides (Fisher Scientific Corp., Fairlawn, NJ) and stored at room temperature until use. The other half of the brain was fixed in 4% PFA for 1 hour at 4°C and then placed into a 20% sucrose for 48 hours at 4°C and then snap-frozen in isopentane. Transverse sections, 10 μm thick, were cut with a cryostat at the fornix region of the corpus callosum, mounted onto gelatin-coated slides, and stored at −70°C until staining. All comparative analyses were focused at the medial region of the corpus callosum that was above the fornix and between the ventricles (corresponding to sections 220–260 of the mouse brain atlas20Sidman RL Abervine JB Pierec ET Atlas of the mouse brain and spinal cord. Harvard University Press, Cambridge1971Google Scholar). The forebrains from another set of mice were removed for RNA analysis. Mid-sagittal sections of the forebrain were frozen immediately on dry ice. All animal procedures were conducted in accordance with guidelines approved by the IACUC and both the University of North Carolina and Columbia University Division of Laboratory Animal Medicine. To identify apoptotic mature oligodendrocytes within the brain, the NeuroTACS assay kit (Trevigen, Gaithersburg, MD) was used to identify apoptotic cells and mature oligodendrocytes were identified by the immunohistochemical detection of the pi isoform of glutathione-S-transferase (GST-pi),21Tansey FA Cammer WP A pi form of glutathione-S-transferase is a myelin- and oligodendrocyte-associated enzyme in mouse brain.J Neurochem. 1991; 57: 95-102Crossref PubMed Scopus (116) Google Scholar which does not detect immature NG2+ oligodendrocyte (unpublished observations). Paraffin-embedded sections were rehydrated in a graded series of alcohol washes, blocked with 5% normal goat serum (NGS)/0.1% Triton for 20 minutes, incubated with 0.1% trypsin/0.5 mol/L Tris for 15 minutes at 37°C, and then incubated with the rabbit polyclonal anti-GST-pi antibody (1:1000; Biotrin, Newton, MA) overnight at 4°C. The tissue sections were then incubated with 1X labeling buffer (Trevigen) for 5 minutes and then incubated with a labeling reaction mixture of 1X labeling buffer, biotin-labeled dNTPs (Trevigen), and Klenow enzyme (Trevigen) for 1.5 hours at 37°C. The reaction was stopped by placing the tissue sections into a 1X stop reaction buffer (Trevigen) for 5 minutes. The tissue sections were then incubated with a fluorescein-conjugated goat anti-rabbit secondary antibody diluted 1:75 in 5% NGS/0.2% Triton/2% streptavidin-Texas red complex (Vector Laboratories, Burlingame, CA) for 1 hour, counterstained with DAPI (Molecular Probes, Eugene, OR), coverslipped, and examined using an Olympus BX60 microscope equipped with epifluorescence optics. To identify oligodendrocyte progenitors, frozen tissue sections were dried, rehydrated, blocked with 5% NGS/0.2% Triton, and then incubated with rabbit anti-NG2 (gift from Dr. William Stallcup, San Diego, CA) diluted 1:400 in 5% NGS/0.2% Triton overnight at 4°C. The sections were then incubated with a fluorescein-conjugated goat anti-rabbit secondary antibody (1:75; Vector Laboratories) for 1 hour, counterstained with DAPI, coverslipped, and examined using an Olympus BX60 microscope equipped with epifluorescence optics. Total RNA from entire half of a forebrain sectioned mid-sagittally along midline was isolated, separated, and transferred to a Zeta-probe nylon membrane as described previously.22Morell P Barrett CV Mason JL Toews AD Hostettler JD Knapp GW Matsushima GK Gene expression in brain during cuprizone-induced demyelination and remyelination.Mol Cell Neurosci. 1998; 12: 220-227Crossref PubMed Scopus (172) Google Scholar Membranes were hybridized with 32P-labeled cDNA probes, synthesized using double-stranded polymerase chain reaction (PCR) methodology23Jansen R Ledley FD Production of high specific activity DNA probes using the polymerase chain reaction.Gene Anal Techn. 1989; 6: 79-83Crossref PubMed Scopus (45) Google Scholar for amplification of cDNA for myelin-associated glycoprotein (MAG).24Lai C Brow MA Nave K-A Noronha AB Quarles RH Bloom FE Milner RJ Sutcliffe JG Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal development, are produced by alternative splicing.Proc Natl Acad Sci USA. 1987; 84: 4337-4341Crossref PubMed Scopus (302) Google Scholar Filters were washed and radioactivity in RNA was quantitated using a Packard Instant Imager electronic autoradiography system. To control for variability in sampling handling, values obtained were normalized to the amount of rRNA in each lane as described previously.22Morell P Barrett CV Mason JL Toews AD Hostettler JD Knapp GW Matsushima GK Gene expression in brain during cuprizone-induced demyelination and remyelination.Mol Cell Neurosci. 1998; 12: 220-227Crossref PubMed Scopus (172) Google Scholar Additional RNA was reverse transcribed into cDNA and then amplified by PCR as described previously.6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar The primer sets for paranodin were 5′-ACAAGGGAGTTACCTGCCATGAAC (sense) and 3′-GCACGGATTGGAGGGAAACG (antisense). The primer sets for G3PDH have been published elsewhere.25Bencsik A Malcus C Akaoka H Giraudon P Belin M-F Bernard A Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression.J Neuroimmunol. 1996; 65: 1-9Abstract Full Text PDF PubMed Scopus (30) Google Scholar Templates were denatured at 95°C for 5 minutes, followed by 30 cycles of denaturation (95°C for 1 minute) primer annealing (62°C for 1.5 minutes) and extension (72°C for 1 minute) with a final extension step at 72°C for 15 minutes. The amplified cDNA mixture of each sample was separated on a 2.5% agarose gel containing ethidium bromide and photographed. The amplified products for paranodin and G3PDH were 696 and 983 bp, respectively. Amplification of the housekeeping gene, G3PDH, confirmed RNA amplification and equal loading of each sample. The mRNA profiles were semiquantitated using a densitometer to normalize all samples for comparison. Microglia/macrophages were identified by binding biotin-conjugated Ricinus communis agglutin-1 (RCA-1; Sigma Chemical Co.) to paraffin-embedded sections and then visualized by the 3′3′-diaminobenzidine methodology (Vector Laboratories) as described previously.19Hiremath MM Saito Y Knapp GW Ting JP-Y Suzuki K Matsushima GK Microglia/macrophage accumulation during cuprizone-induced demyelination in C57BL/6 mice.J Neuroimmunol. 1998; 92: 38-49Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar Transgenic C57BL6 mice, in which green fluorescent protein expression is driven by the chicken β-actin promoter (GFP tg mice), were purchased from Jackson Laboratories. Progenitors were isolated from the corpus callosum in 8-week-old GFP tg mice as described previously.26Mason JL Goldman JE A2B5+ and O4+ cycling progenitors in the adult forebrain white matter respond differentially to PDGF-AA, FGF-2 and IGF-1.Mol Cell Neurosci. 2002; 20: 30-42Crossref PubMed Scopus (83) Google Scholar, 27Gensert JM Goldman JE Heterogeneity of cycling progenitors in the adult mammalian cortex and white matter.J Neurobiol. 2001; 48: 75-86Crossref PubMed Scopus (83) Google Scholar Briefly, the mice were anesthetized and the corpus callosum was carefully dissected and then mechanically and enzymatically dissociated with 0.125% trypsin (Sigma Chemical Co.) and 0.02% collagenase (Worthington Biochemical Co., Lakewood, NJ). The cellular suspension was filtered through sterile 70-μm mesh and the trypsin neutralized with 10% heat-inactivated serum. Cells were collected by centrifugation and then resuspended in 0.9 mol/L sucrose/MEM (Gibco, Carlsbad, CA). This cellular suspension was centrifuged and the progenitors (bottom layer) were collected and resuspended in serum-free medium (see below). Approximately 5 × 105 cells were isolated from the corpus callosum of each adult mouse with the cells being pooled from three corpora collosa. O4+ progenitors were then purified from the total progenitors using immunopanning methods described previously.26Mason JL Goldman JE A2B5+ and O4+ cycling progenitors in the adult forebrain white matter respond differentially to PDGF-AA, FGF-2 and IGF-1.Mol Cell Neurosci. 2002; 20: 30-42Crossref PubMed Scopus (83) Google Scholar Briefly, immunopan plates were created by incubating goat anti-mouse IgM antibody (20 μg/ml; Sigma Chemical Co.), diluted in 50 mmol/L Tris, in Falcon 1007 petri dishes (Fisher) overnight at 37°C. The plates were then washed, incubated with O4 hybridoma supernatant (undiluted) at 37°C for 3 hours, and then blocked with 10% normal goat serum (NGS)/10% bovine serum albumin (BSA) at 37°C for 30 minutes. 5 × 105 cells were then incubated in each O4 immunoplate for 1 hour at 37°C. The plates were then washed and coated with trypsin/EDTA for 10 minutes at 37°C. The detached cells were collected in an equal volume of 10% heat-inactivated serum to neutralize the trypsin, centrifuged, and then resuspended in serum-free medium (1 × 104 O4+ cells/μl). Approximately 3.1 × 105 O4+ cells were isolated from the three corpora collosa. Serum-free medium was composed of d-biotin (10 ng/ml; Sigma Chemical Co.), insulin (5 μg/ml; Sigma Chemical Co.), progesterone (20 nM; Sigma Chemical Co.), putrescine (100 μmol/L; Sigma Chemical Co.), selenium (5 ng/ml; Sigma Chemical Co.), transferrin (50 μg/ml; Sigma Chemical Co.), Hepes buffer (15 mmol/L; Sigma Chemical Co.), 3,3,5-triiodo-l-thyronine (15 nM; Sigma Chemical Co.), penicillin/streptomycin (100U/100 μg/ml; Gibco) and BSA (1 mg/ml; Sigma Chemical Co.) in DMEM/F12 (Gibco). At 8 weeks of age, wild-type male recipient mice (Jackson Laboratories) were fed a diet of milled Purina mouse chow containing 0.2% cuprizone by weight for 12 weeks to induce chronic demyelination within the corpus callosum. The mice were then anesthetized with ketamine (80 mg/kg body weight; Henry Schein Pharmaceutical, Port Washington, NY) and xylazine (10 mg/kg body weight; Henry Schein Pharmaceutical) and positioned in an immobilization apparatus designed for stereotactic surgery (David Kopf Instruments, Tujunga, CA). The scalp was opened and the O4+ oligodendrocyte progenitors isolated from the corpus callosum in GFP tg donor mice were injected (1 × 104 O4+ cells in 1 μl of serum-free medium) unilaterally into the chronically demyelinated corpus callosum in the wild-type recipient mice with a 10-μl Hamilton syringe using stereotactic coordinates of 0.7 mm posterior and 0.3 mm lateral to bregma at a depth of 1.7 mm.28Franklin KBJ Paxinos G The mouse brain in stereotaxic coordinates. Academic Press, San Diego1997Google Scholar Additional control recipient mice were stereotactically injected with serum-free medium containing no progenitors. The opening in the skull was then filled with Gelfoam, the area swabbed with penicillin and streptomycin (Gibco) and the wound was sutured. Immediately after transplantation, the recipient mice were returned to a normal diet for 4 weeks allowing recovery. The mice were then anesthetized, perfused through the heart with either 4% PFA (light microscopy) or 2.5% glutaraldehyde (electron microscopy) and processed for analysis as described previously.6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar, 18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar All animal procedures were conducted in accordance with guidelines approved by the IACUC and the Columbia University Division of Laboratory Animal Medicine. For light microscopy, frozen tissue sections were dried, rehydrated, blocked with 5% NGS/0.2% Triton and then incubated with the rabbit polyclonal anti-GST-pi antibody (1:1000; Biotrin) and the mouse monoclonal anti-GFP antibody (1:500; Chemicon, Temecula, CA) overnight at 4°C. The sections were then incubated with a biotin-conjugated goat anti-mouse secondary antibody (1:100; Vector Laboratories) and a rhodamine-conjugated goat anti-rabbit antibody (1:100; Southern Biotechnology Associates) for 1 hour. The sections were then incubated with a 2% streptavidin-fluorescein complex (Vector) in 5% NGS/0.2% Triton, counterstained with DAPI, coverslipped, and examined using an Olympus BX60 microscope equipped with epifluorescence optics. For electron microscopy, glutaraldehyde-fixed brain samples were prepared as described previously.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar Coronal sections (1 μm) were stained with toluidine blue and the medial region of the corpus callosum was identified by light microscopy. The tissue was then trimmed and reoriented for thin sectioning so that cross sections of the corpus callosum could be examined by electron microscopy. Thin sections were cut, stained with uranyl acetate and lead citrate, photographed, and then the electron micrographs were analyzed as described previously.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google Scholar One thousand fibers (≥0.3 μm in diameter) from five different random sections of the corpus callosum were examined from each mouse (three mice at each time point). Immunopositive cells were obtained by counting only those cells with an identified nucleus, observable by DAPI staining, within the medial region of the corpus callosum. Individual cell counts were conducted on both sides of midline corresponding to an area of 0.033 mm2. The cell counts from both areas were averaged to give a total for each tissue section. The cell counts (number of cells/mm2) are presented in the text as the mean and SEM from at least three mice for each time point. Statistical comparisons were made using a one-factor between-subjects analysis of variance. Multiple comparisons between groups were made using Tukey's test. Cuprizone toxicity markedly depleted the mature oligodendrocyte population, as detected by immunocytochemistry using the anti-GST-pi antibody (Figure 1, Figure 2).6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar When mice were returned to a normal diet after 6 weeks of cuprizone feeding, the oligodendrocyte population returned to control numbers (Figure 1, Figure 2).6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar In contrast, in mice that were maintained on a cuprizone diet for up to 12 weeks, a large number of oligodendrocytes reappeared (Figure 1D), although numbers never fully returned to control (Figure 2A). Despite the presence of oligodendrocytes, however, there was little remyelination.18Mason JL Langaman C Morell P Suzuki K Matsushima GK Episodic demyelination and subsequent remyelination within the murine central nervous system: changes in axonal calibre.Neuropathol Appl Neurobiol. 2001; 27: 50-58Crossref PubMed Scopus (138) Google ScholarFigure 2Cuprizone induces the apoptotic death of mature oligodendrocytes and the progressive depletion of the oligodendrocyte progenitor population. The total number of mature oligodendrocytes, the number of non-apoptotic mature oligodendrocytes (black bars) and the number apoptotic mature oligodendrocytes (white bars) were quantitated and plotted as mean ± SEM for triplicate determinations (A, B). The number of GST-pi+ and apoptotic GST-pi+ cells/mm2 in mice fed cuprizone continuously for 12 weeks (A) and in mice fed cuprizone for 6 weeks and then returned to a normal diet for 6 weeks (B) are shown. C: The total number of NG2+ oligodendrocyte progenitors within the medial region of the corpus callosum were also quantitated and plotted as mean ± SEM for triplicate determinations in mice fed cuprizone continuously for 12 weeks (white bars) and in mice fed cuprizone for 6 weeks and then returned to a normal diet for 6 weeks (black bars). *P < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The inability of the oligodendrocyte population to regenerate adequately and remyelinate the axons during long-term cuprizone treatment could be a consequence of the newly regenerated cells undergoing continued apoptosis as observed with the oligodendrocytes in acute lesions (Figure 2A).6Mason JL Jones JJ Taniike M Morell P Matsushima GK Mature oligodendrocyte apoptosis precedes IGF-1 production and oligodendrocyte progenitor accumulation and differentiation during demyelination/remyelination.J Neurosci Res. 2000; 61: 251-262Crossref PubMed Scopus (180) Google Scholar To address this issue, we used immunohistochemistry to determine whether apoptotic (TUNEL ass
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