Stimulation of Adult Oligodendrogenesis by Myelin-Specific T Cells
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.006
ISSN1525-2191
AutoresHelle Hvilsted Nielsen, Henrik Toft–Hansen, Kate Lykke Lambertsen, Trevor Owens, Bente Finsen,
Tópico(s)Immune Response and Inflammation
ResumoIn multiple sclerosis (MS), myelin-specific T cells are normally associated with destruction of myelin and axonal damage. However, in acute MS plaque, remyelination occurs concurrent with T-cell infiltration, which raises the question of whether T cells might stimulate myelin repair. We investigated the effect of myelin-specific T cells on oligodendrocyte formation at sites of axonal damage in the mouse hippocampal dentate gyrus. Infiltrating T cells specific for myelin proteolipid protein stimulated proliferation of chondroitin sulfate NG2–expressing oligodendrocyte precursor cells early after induction via axonal transection, resulting in a 25% increase in the numbers of oligodendrocytes. In contrast, T cells specific for ovalbumin did not stimulate the formation of new oligodendrocytes. In addition, infiltration of myelin-specific T cells enhanced the sprouting response of calretinergic associational/commissural fibers within the dentate gyrus. These results have implications for the perception of MS pathogenesis because they show that infiltrating myelin-specific T cells can stimulate oligodendrogenesis in the adult central nervous system. In multiple sclerosis (MS), myelin-specific T cells are normally associated with destruction of myelin and axonal damage. However, in acute MS plaque, remyelination occurs concurrent with T-cell infiltration, which raises the question of whether T cells might stimulate myelin repair. We investigated the effect of myelin-specific T cells on oligodendrocyte formation at sites of axonal damage in the mouse hippocampal dentate gyrus. Infiltrating T cells specific for myelin proteolipid protein stimulated proliferation of chondroitin sulfate NG2–expressing oligodendrocyte precursor cells early after induction via axonal transection, resulting in a 25% increase in the numbers of oligodendrocytes. In contrast, T cells specific for ovalbumin did not stimulate the formation of new oligodendrocytes. In addition, infiltration of myelin-specific T cells enhanced the sprouting response of calretinergic associational/commissural fibers within the dentate gyrus. These results have implications for the perception of MS pathogenesis because they show that infiltrating myelin-specific T cells can stimulate oligodendrogenesis in the adult central nervous system. T cell infiltration, demyelination, and axonal damage are central pathologic features of multiple sclerosis (MS). Whereas the primary immune attack on oligodendrocytes and myelin is effected by T cells,1Weiner H.L. Multiple sclerosis is an inflammatory T-cell–mediated autoimmune disease.Arch Neurol. 2004; 61: 1613-1615Crossref PubMed Scopus (203) Google Scholar, 2Lucchinetti C. Brück W. Parisi J. Scheithauer B. Rodriguez M. Lassmann H. 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Phenotypic changes in NG2+ cells after spinal cord injury.J Neurotrauma. 2006; 23: 1726-1738Crossref PubMed Scopus (24) Google Scholar axonal lesions,13Dehn D. Burbach G.J. Schafer R. Deller T. NG2 upregulation in the denervated rat fascia dentata following unilateral entorhinal cortex lesion.Glia. 2006; 53: 491-500Crossref PubMed Scopus (26) Google Scholar, 14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar and inflammation.15Schonberg D.L. Popovich P.G. McTigue D.M. Oligodendrocyte generation is differentially influenced by toll-like receptor (TLR) 2 and TLR4-mediated intraspinal macrophage activation.J Neuropathol Exp Neurol. 2007; 66: 1124-1135Crossref PubMed Scopus (84) Google Scholar During differentiation, OPCs down-regulate NG2 as cells acquire markers of mature oligodendrocytes such as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP).16Dawson M.R. Levine J.M. Reynolds R. NG2-expressing cells in the central nervous system: are they oligodendroglial progenitors?.J Neurosci Res. 2000; 61: 471-479Crossref PubMed Scopus (344) Google Scholar The axonal damage that occurs within and distal to the acute MS lesion can be modeled in the hippocampal dentate gyrus by transection of the perforant pathway (PP), resulting in degeneration of the PP axons and their myelin sheaths in the outer part of the molecular layer.17Jensen M.B. Gonzalez B. Castellano B. Zimmer J. Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.Exp Brain Res. 1994; 98: 245-260Crossref PubMed Scopus (123) Google Scholar, 18Kwidzinski E. Mutlu L.K. Kovac A.D. Bunse J. Goldmann J. Mahlo J. Aktas O. Zipp F. Kamradt T. Nitsch R. Bechmann I. Self-tolerance in the immune privileged CNS: lessons from the entorhinal cortex lesion model.J Neural Transm Suppl. 2003; (–4929)PubMed Google Scholar, 19Nielsen H.H. Ladeby R. Fenger C. Toft-Hansen H. Babcock A.A. Owens T. Finsen B. Enhanced microglia clearance of myelin debris in T-cell infiltrated central nervous system.J Neuropathol Exp Neurol. 2009; 68: 845-856Crossref PubMed Scopus (12) Google Scholar PP lesions also induce proliferation of OPCs, which results in formation of new oligodendrocytes.14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar These newly formed oligodendrocytes are presumed to myelinate the axonal sprouts that extend from other afferent fiber systems in the dentate gyrus20Phinney A.L. Calhoun M.E. Woods A.G. Deller T. Jucker M. Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice.Eur J Neurosci. 2004; 19: 1731-1740Crossref PubMed Scopus (15) Google Scholar, 21Steward O. Vinsant S.L. The process of reinnervation in the dentate gyrus of adult rats: a quantitative electron microscopic analysis of terminal proliferation and reactive synaptogenesis.J Comp Neurol. 1983; 214: 370-386Crossref Scopus (232) Google Scholar such as the associational/commissural afferents from the calretinergic hilar mossy cells.20Phinney A.L. Calhoun M.E. Woods A.G. Deller T. Jucker M. Stereological analysis of the reorganization of the dentate gyrus following entorhinal cortex lesion in mice.Eur J Neurosci. 2004; 19: 1731-1740Crossref PubMed Scopus (15) Google Scholar, 22Blasco-Ibanez J.M. Freund T.F. Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus.Hippocampus. 1997; 7: 307-320Crossref PubMed Scopus (130) Google Scholar, 23Del Turco D. Woods A.G. Gebhardt C. Phinney A.L. Jucker M. Frotscher M. Deller T. Comparison of commissural sprouting in the mouse and rat fascia dentata after entorhinal cortex lesion.Hippocampus. 2003; 13: 685-699Crossref PubMed Scopus (37) Google Scholar Indeed, in stratum radiatum of the hippocampal CA3 region, lesion-induced axonal sprouting is associated with formation of more oligodendrocytes and more myelin.24Drojdahl N. Nielsen H.H. Gardi J.E. Wree A. Peterson A.C. Nyengaard J.R. Eyer J. Finsen B. Axonal plasticity elicits long-term changes in oligodendroglia and myelinated fibers.Glia. 2010; 58: 29-42Crossref PubMed Scopus (18) Google Scholar Because remyelination ultimately fails in MS,25Patrikios P. Stadelmann C. Kutzelnigg A. Rauschka H. Schmidbauer M. Laursen H. Sorensen P.S. Bruck W. Lucchinetti C. Lassmann H. Remyelination is extensive in a subset of multiple sclerosis patients.Brain. 2006; 129: 3165-3172Crossref PubMed Scopus (569) Google Scholar it is assumed that autoimmune demyelination reduces the capacity for myelin repair.26Ludwin S.K. Chronic demyelination inhibits remyelination in the central nervous system: an analysis of contributing factors.Lab Invest. 1980; 43: 382-387PubMed Google Scholar, 27Niehaus A. Shi J. Grzenkowski M. Diers-Fenger M. Archelos J. Hartung H.P. Toyka K. Bruck W. Trotter J. Patients with active relapsing-remitting multiple sclerosis synthesize antibodies recognizing oligodendrocyte progenitor cell surface protein: implications for remyelination.Ann Neurol. 2000; 48: 362-371Crossref PubMed Scopus (102) Google Scholar We investigated the effect of myelin-specific T cells on the formation of oligodendrocytes in the dentate gyrus of mice subjected to PP transection. Via adoptive transfer of T cells specific for myelin proteolipid protein (PLP) before axonal lesioning, infiltration of T cells into the dentate gyrus was significantly enhanced, compared with limited T-cell infiltration in PP-lesioned mice with adoptive transfer of ovalbumin (OVA)–specific T cells or lesioned naïve mice. A significantly higher increase in the number of postproliferative oligodendrocytes was observed in the PP-lesioned TPLP-recipient mice than in PP-lesioned TOVA-recipient and naïve mice. Furthermore, the increased oligodendrogenesis was preceded by increased proliferation of NG2+ OPCs in the dentate gyrus. These changes correlated with an increased clearance of myelin debris and increased sprouting of calretinergic associational/commissural fibers. Our results demonstrate that myelin-specific T cells can stimulate oligodendrogenesis in vivo. Female SJL mice aged 8 to 10 weeks were obtained from Taconic Europe A/S (Ejby, Denmark) and maintained in a pathogen-free temperature- and humidity-controlled environment with a 12-hour light-dark cycle, and were provided with food and water ad libitum. Experiments were approved by the National Danish Animal Care Committee (J.nr. 192000/561-272 and J.nr. 192000/51-272). TPLP and TOVA were generated as previously described.28Krakowski M.L. Owens T. Naïve T lymphocytes traffic to inflamed central nervous system, but require antigen recognition for activation.Eur J Immunol. 2000; 30: 1002-1009Crossref PubMed Scopus (87) Google Scholar Donor mice were immunized via two s.c. injections, at the base of the tail and in the flank, with 50 μL emulsion of Mycobacterium tuberculosis H37 RA (2 mg/mL) (Difco Laboratories, Inc., Detroit, MI) in incomplete Freund's adjuvant solution (Difco Laboratories, Inc.) and PLP139–151 (1 mg/mL) (KJ Ross-Petersen ApS, Klampenborg, Denmark) or ovalbumin (30 mg/mL) (Sigma-Aldrich Corp., St. Louis, MO). Lymph nodes were collected on day 11, and cells were cultured for 4 days in RPMI-1640 medium (Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen Corp.), 2 mmol/L l-glutamine (Sigma-Aldrich Corp.), 50 μmol/L 2-mercaptoethanol (Bie & Berntsen A/D, Herlev, Denmark), and 5 μg/mL PLP. Proliferation was measured using the Vybrant MTT Cell Proliferation Assay Kit (Invitrogen Corp.). TPLP and TOVA cultures showed equal proliferation rates before cells were collected on a Ficoll-Hypaque gradient (Amersham Pharmacia Biotech, Inc., Piscataway, NJ), counted, and injected i.v. into recipient mice (6 × 106 blasts per mouse or 28% to 30% of the cells injected). TPLP- and TOVA-recipient mice (TPLP and TOVA mice, respectively) were weighed and clinically evaluated daily. TOVA mice demonstrated no symptoms. TPLP mice reached experimental allergic encephalomyelitis (EAE) grade 0 to 2 before termination at 7 days post lesion (11 days post transfer). For studies of cytokine expression of T cells in vitro, TPLP and TOVA cells were generated, cultivated, and harvested as described above, transferred into TRIzol (Invitrogen Corp.), and stored at −80°C. Lymph node cells from naïve mice served as controls. Anterograde axonal degeneration was induced via stereotactic transection of the PP using a wire knife.14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar Mice were PP lesioned at 4 days post transfer, when myelin-specific T cells usually enter the CNS after i.v. injection.29Flugel A. Berkowicz T. Ritter T. Labeur M. Jenne D.E. Li Z. Ellwart J.W. Willem M. Lassmann H. Wekerle H. Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis.Immunity. 2001; 14: 547-560Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar Mice were euthanized at 2 days post lesion (11 TPLP, 8 TOVA, and 10 naïve) and 7 days post lesion (7 TPLP, 8 TOVA, and 6 naïve). Unlesioned TOVA and TPLP mice (n = 6 and 8, respectively) were euthanized at 11 days post transfer, when TPLP mice demonstrated symptoms of EAE grade 0 to 2. Unmanipulated mice (n = 6) and unlesioned the contralateral dentate gyrus served as controls. To study the cytokine profile using quantitative PCR (qPCR), a similar set of animals was generated (n = 8 to 12 per group). For investigations of cellular proliferation and differentiation, animals received a i.p. bolus injection of BrdU (5′-bromo-2′-deoxyuridine) dissolved in isotonic saline solution (50 mg/kg) at 2 days post lesion. Animals euthanized at day 2 received the injection 1 hour before sacrifice. Mice were deeply anesthetized using 0.05 mL pentobarbital (200 mg/mL) and perfused through the left ventricle using 5 mL 0.15 mmol/L Sørensen phosphate buffer (pH 7.4) followed by 20 mL 4% paraformaldehyde in 0.15 mmol/L Sørensen phosphate buffer (pH 7.4). The brains were postfixed in 4% paraformaldehyde for 1½ hours, immersed in 20% sucrose overnight, frozen, and serially cut into 16-μm parallel cryostat sections. For qPCR, animals were perfused using 20 mL 0.15 mmol/L Sørensen phosphate buffer, and the ipsilateral hippocampus was dissected and stored in TRIzol at −80°C. For in situ hybridization, PP-lesioned TPLP, TOVA, and naïve mice (n = 3 or 4 per group) were decapitated, and the brains were frozen, cut as above, and stored at −80°C. The quality of the lesion was controlled using Fluoro-Jade staining.30Dissing-Olesen L. Ladeby R. Nielsen H.H. Toft-Hansen H. Dalmau I. Finsen B. Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse.Neuroscience. 2007; 149: 112-122Crossref PubMed Scopus (49) Google Scholar Only mice that demonstrated a dense band of green fluorescence in the outer molecular layer, reflecting complete transection of both the medial and lateral PP, were included in the study.30Dissing-Olesen L. Ladeby R. Nielsen H.H. Toft-Hansen H. Dalmau I. Finsen B. Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse.Neuroscience. 2007; 149: 112-122Crossref PubMed Scopus (49) Google Scholar, 31Matthews D.A. Cotman C. Lynch G. An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. I: Magnitude and time course of degeneration.Brain Res. 1976; 115: 1-21Crossref PubMed Scopus (384) Google Scholar Neurofilament (NF) was detected by using primary monoclonal rat anti-mouse phosphorylated NF antibody (MAB5448; dilution 1:100; Chemicon International, Inc., Temecula, CA), secondary biotinylated species-specific monoclonal goat anti-rat antibody (RPN 1005; dilution 1:200; Amersham Pharmacia Biotech, Inc.), and streptavidin-horseradish peroxidase (P397; dilution 1:200; Dako A/S, Glostrup, Denmark), as described by Nielsen et al.14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar T cells were visualized as described for NF using monoclonal rat anti-human CD3 antibody (MCA1477; dilution 1:200; AbD Serotec, Ltd., Kidlington, Oxfordshire, England). Cross-reactivity for murine CD3 was confirmed via location of CD3+ cells to the periarteriolar sheath in murine spleen sections. OPCs were visualized using polyclonal rabbit raised against chondroitin sulfate proteoglycan NG2 (AB5320; dilution 1:500; Chemicon International, Inc.). Oligodendrocytes and myelin were visualized using monoclonal mouse anti-rat Rip antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) recognizing 2′, 3′-cyclic nucleotide 3′-phosphodiesterase.32Friedman B. Hockfield S. Black J.A. Woodruff K.A. Waxman S.G. In situ demonstration of mature oligodendrocytes and their processes: an immunocytochemical study with a new monoclonal antibody, Rip.Glia. 1989; 2: 380-390Crossref PubMed Scopus (234) Google Scholar, 33Watanabe M. Sakurai Y. Ichinose T. Aikawa Y. Kotani M. Itoh K. Monoclonal antibody Rip specifically recognizes 2′,3′-cyclic nucleotide 3′-phosphodiesterase in oligodendrocytes.J Neurosci Res. 2006; 84: 525-533Crossref PubMed Scopus (52) Google Scholar The antibody was biotinylated by Karsten Skjødt, University of Southern Denmark (Odense) and used at a concentration of 0.05 mg/mL. NG2 and CNP stainings were counterstained using toluidine blue to facilitate cell counting. Calretinergic fibers were visualized using polyclonal rabbit anti-calretinin antibody (7699/3H; dilution 1:10.000; Swant, Inc., Marly, Switzerland) diluted in 10% fetal bovine serum containing 1% triton. Rinses were extended to 3 × 2 hours. Primary and secondary antibodies were incubated overnight at 4°C. After staining for NG2 or CNP, sections were rinsed in 2× standard saline citrate solution [300 mmol/L NaCl and 30 mmol/L and sodium citrate (pH 8.0)] for 2 × 15 minutes at room temperature, incubated in a 49% solution of formamide in 2× standard saline citrate solution for 2 hours at 60°C, rinsed in 2× standard saline citrate solution for 2 × 5 minutes at 60°C, and incubated in 2N HCl in 0.05 mmol/L Tris-buffered saline solution for 30 minutes at 37°C. Sections were rinsed and incubated using monoclonal rat anti-BrdU antibody (AB6326; dilution 1:100; Abcam Ltd., Cambridge, England), which was detected using biotinylated goat anti-rat Ig antibody (RPN 1005; dilution 1:200; Amersham Pharmacia Biotech, Inc.), and alkaline phosphatase–conjugated streptavidin (P396; Dako A/S), and incubated in an alkaline phosphatase developer as previously described.34Fenger C. Drojdahl N. Wirenfeldt M. Sylvest L. Jorgensen O.S. Meldgaard M. Lambertsen K.L. Finsen B. Tumor necrosis factor and its p55 and p75 receptors are not required for axonal lesion-induced microgliosis in mouse fascia dentata.Glia. 2006; 54: 591-605Crossref PubMed Scopus (20) Google Scholar Calretinergic fibers were detected using Alexa 488–conjugated sodium arsenite (S-32354; dilution 1:500; Invitrogen Corp.) along with fluorescence detection of myelin basic protein (MBP) using Alexa 594–conjugated goat anti-rabbit Ig (A-11012; dilution 1:500; Invitrogen Corp.) as described by Nielsen et al.35Nielsen H.H. Ladeby R. Fenger C. Toft-Hansen H. Babcock A.A. Owens T. Finsen B. Enhanced microglial clearance of myelin debris in T cell–infiltrated central nervous system.J Neuropathol Exp Neurol. 2009; 68: 845-856Crossref PubMed Scopus (31) Google Scholar The cellular nuclei were visualized using the nucleic acid stain DAPI (D3571; Invitrogen Corp.), which was added in a concentration of 300 nmol/L to the Tris-buffered saline solution during the last rinse. Non-specific staining was controlled for by incubation without the primary antibody, with an isotype-specific control (rat IgG1 or rat IgG2b; Nordic Biosite AB, Täby, Sweden) or with rabbit (X902) or goat (X907) serum (both from Dako A/S), and exhibited no staining. A mixture of two alkaline phosphatase–labeled DNA probes (DNA Technology A/S, Risskov, Denmark) complementary to bases 169 to 196 (5′-GGCTTTCAATGACTGTGCCGTGGCAGTA-3′) and 530 to 557 (5′-CGCTTCCTGAGGCTGGATTCCGGCAACA-3′) was used for detection of interferon-γ (IFN-γ) cDNA. Probe specificity was confirmed by hybridizing with each probe alone or with a probe mixture, showing identical regional and cellular localization of the in situ signal but with a stronger signal in sections hybridized with the probe mixture. In addition, sections pretreated with RNase A (27-0323-01; Amersham Pharmacia Biotech, Inc.) before hybridization or hybridized using a 100-fold excess of unlabeled IFN-γ probe mixture or buffer alone were devoid of signal (see Supplemental Figure S1B at http://ajp.amjpathol.org). RNA extraction, cDNA synthesis, and qPCR were performed on dissected hippocampi36Meldgaard M. Fenger C. Lambertsen K.L. Pedersen M.D. Ladeby R. Finsen B. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (43) Google Scholar using a sequence detection system (PRISM 7300; Applied Biosystems, Inc., Foster City, CA) and using either a Taqman probe (TAG Copenhagen A/S, Copenhagen, Denmark) or SYBR Green (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) as fluorescent reporter molecules. A standard curve was prepared from a fivefold dilution series of cDNA from spinal cord obtained from animals with symptoms of EAE. Primers and probes for tumor necrosis factor (TNF)–α and IFN-γ and the ε-chain of the CD3 complex were used as previously described.34Fenger C. Drojdahl N. Wirenfeldt M. Sylvest L. Jorgensen O.S. Meldgaard M. Lambertsen K.L. Finsen B. Tumor necrosis factor and its p55 and p75 receptors are not required for axonal lesion-induced microgliosis in mouse fascia dentata.Glia. 2006; 54: 591-605Crossref PubMed Scopus (20) Google Scholar, 37Lambertsen K.L. Gregersen R. Meldgaard M. Clausen B.H. Heibol E.K. Ladeby R. Knudsen J. Frandsen A. Owens T. Finsen B. A role for interferon-gamma in focal cerebral ischemia in mice.J Neuropathol Exp Neurol. 2004; 63: 942-955PubMed Google Scholar For IL-4, we used forward primer 5′-AAACATGGGAAAACTCCA-3′ and reverse primer 5′-CAGCTTATCGATGAATCCA-3′); for IL-10, forward primer 5′-AGGACTTTAAGGGTTACT-3′ and reverse primer 5′-AATGCTCCTTGATTTCTG-3′; for IL-17, forward primer 5′-GCTTCATCTGTGTCTCTG-3′ and reverse primer 5′-GAACGGTTGAGGTAGTCT-3′). For NG2, we used forward primer 5′-TCCCGGAGAGAGGTGGAAGAG-3′ and reverse primer 5′-GGTCCATCTCTGAGGCATTAGC, and probe 5′-AAGGCGTCTGTCTGTGTCTCACTTCCATCA-3′. For insulin-like growth factor-1 (IGF-1), we used forward primer 5′-CCGAGGGGC TTTTACTTCAACAA-3′ and reverse primer 5′-CGGAAGCAACACTCATCCACAA-3′. For brain-derived neurotrophic factor (BDNF), we used forward primer 5′-GGCCCAACGAAGAAAACCAT-3′ and reverse primer 5′-AGCATCACCCGGGAAGTGT-3′. Specificity of the PCR primer/probe set was validated as described in Meldgaard et al.36Meldgaard M. Fenger C. Lambertsen K.L. Pedersen M.D. Ladeby R. Finsen B. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (43) Google Scholar Test gene data were normalized to the reference gene hypoxanthine phosphoribosyltransferase 1 and calibrated to a pool of control samples from animals not operated on. mRNA expression was normalized to the expression of unmanipulated controls, which was set to 1.0. IFN-γ, IL-4, IL-17, and BDNF mRNA expressions were normalized to the expression in unlesioned TOVA mice because we were unable to detect IFN-γ, IL-4, IL-17, or BDNF mRNA from these cytokines in the hippocampus of unlesioned naïve mice. In cases in which mRNA was undetectable (more than 40 cycles or undetermined), the fold increase was set at zero. Changes in mRNA level less than twofold were not subjected to statistical analysis.36Meldgaard M. Fenger C. Lambertsen K.L. Pedersen M.D. Ladeby R. Finsen B. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (43) Google Scholar Cell counting in the molecular layer of the temporal dentate gyrus of PP-lesioned mice was performed using the computer-assisted cast-grid microscope system (CAST2; Visiopharm A/S, Hoersholm, Denmark). The CD3+ cells were defined by their brown plasmalemma. NG2+ cells were defined by a toluidine blue–stained nucleus surrounded by a brown plasmalemma from which more than one process extended. CNP+ cells were defined by a toluidine blue–stained nucleus surrounded by a brown plasmalemma. BrdU+NG2+ cells and BrdU+CNP+ cells were defined as NG2+ and CNP+ cells with a bluish black nucleus. To count the cells using the method of Nielsen et al.,14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar cells were counted in the molecular layer of the dentate gyrus in 10 parallel sections 160 μm apart using a counting frame of 4900 μm2 (fraction of total area, 61%) along the rostrocaudal axis of the hippocampus, starting in the section in which the dentate gyrus first demonstrated a typical U shape (−1900 μm from the bregma), as previously reported.14Nielsen H.H. Ladeby R. Drojdahl N. Peterson A.C. Finsen B. Axonal degeneration stimulates the formation of NG2+ cells and oligodendrocytes in the mouse.Glia. 2006; 54: 105-115Crossref PubMed Scopus (29) Google Scholar At least 150 to 200 single-labeled NG2+ or CNP+ cells were counted per animal. To account for potential differences in the quality of the stainings, cell numbers are reported as the ratio between cell numbers obtained from the deafferented ipsilateral molecular layer compared with cell numbers obtained from the contralateral molecular layer (NG2i/c and CNPi/c). Because of the virtual absence of T cells in the contralateral molecular layer, the total number of counted CD3+ T cells is given. To estimate the area of the inner and outer molecular layers, calretinin-stained sections were analyzed using the CAST2 system. The number of points hitting the inner and outer molecular layers (P) was counted on sections with a mean distance (t) of 160 μm. The sum of the areas of various brain sections was calculated by multiplying the total number of counted points (P) with the computer-given factor of area per point (Apoint = 6.8582 mm2). The total area (Atotal) was calculated using the formula Atotal = ΣP × Apoint × t. Results are given as the ratio between the area of the inner molecular layer and the entire molecular layer. Results are given as mean ± SD. Comparison of medians in two groups was performed using the Mann-Whitney rank sum test. For multiple comparisons, Kruskal-Wallis one-way analysis of variance was performed, followed by Dunn's multiple comparisons test. Statistical analyses were performed using Prism 4.0b software for Macintosh (GraphPad Software, Inc., San Diego, CA). P values are indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. Staining for NF directly visualized the PP fibers terminating as a broad band of NF+ fibers in the outer molecular layer (Figure 1, A and C). As expected, this band of NF+ fibers was completely obliterated by transection of the PP, which focused attention on the NF+ fiber systems that persisted in the inner and outer molecular layers of the dentate gyrus after PP lesioning (Figure 1, B and D). Furthermore, sections from all PP-lesioned TPLP, TOVA, and naïve mice were stained with Fluoro-Jade for visualization of the area of degenerating axons and terminals. As expected, Fluoro-Jade staining demonstrated a sharply demarcated band of green fluorescence in the outer part of the molecular layer of the
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