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• W2077896051 abstract "To examine the role of tumor necrosis factor (TNF)-α in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-α was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-α fusion gene. In two independent GFAP-TNFα transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25−) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of α- and β-chemokines was induced or up-regulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFα mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFα mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-α in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury. To examine the role of tumor necrosis factor (TNF)-α in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-α was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-α fusion gene. In two independent GFAP-TNFα transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25−) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of α- and β-chemokines was induced or up-regulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFα mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFα mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-α in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury. Tumor necrosis factor (TNF)-α is a multifunctional pro-inflammatory cytokine pivotal in the regulation of the host response during infection and inflammation and is also implicated in the pathogenesis of many autoimmune diseases.1Fiers W Tumor necrosis factor: characterization at the molecular, cellular and in vivo level.FEBS Lett. 1991; 285: 199-212Crossref PubMed Scopus (679) Google Scholar, 2Grunfeld C Palladino Jr, MA Tumor necrosis factor: immunologic, antitumor, metabolic and cardiovascular activities.Adv Intern Med. 1990; 35: 45-72PubMed Google Scholar, 3Beutler B Grau GE Tumor necrosis factor in the pathogenesis of infectious diseases.Crit Care Med. 1993; 21: S423-S435Crossref PubMed Scopus (221) Google Scholar A convincing body of evidence implicates TNF-α in the pathogenesis of inflammation in the central nervous system (CNS), including multiple sclerosis (MS),4Raine CS Multiple sclerosis: TNF revisited with promise.Nature Med. 1995; 1: 211-214Crossref PubMed Scopus (90) Google Scholar, 5Merrill JE Proinflammatory and antiinflammatory cytokines in multiple sclerosis and central nervous system acquired immunodeficiency syndrome.J Immunol. 1992; 12: 167-170Google Scholar stroke,6Feuerstein GZ Liu T Barone FC Cytokines, inflammation, and brain injury: role of tumor necrosis factor-α.Cerebrovasc Brain Metab Rev. 1994; 6: 341-360PubMed Google Scholar and infectious diseases ranging from cerebral malaria7Grau GE Modlin RL Immune mechanisms in bacterial and parasitic diseases: protective immunity versus pathology.Curr Opin Immunol. 1991; 3: 480-485Crossref PubMed Scopus (14) Google Scholar and bacterial meningitis8Quagliarello V Scheld WM Bacterial meningitis: pathogenesis, pathophysiology, and progress.N Engl J Med. 1992; 327: 864-872Crossref PubMed Scopus (404) Google Scholar to HIV encephalopathy.9Griffin DE Cytokines in the brain during viral infection: clues from HIV-associated dementia.J Clin Invest. 1997; 100: 2948-2951Crossref PubMed Scopus (99) Google Scholar The sources for TNF-α production in these pathological states may be quite varied, and in addition to infiltrating leukocytes such as macrophages and T cells, significant local production of this cytokine may also come from astrocytes,10Chung IY Benveniste EN Tumor necrosis factor-α production by astrocytes.J Immunol. 1990; 144: 2999-3007PubMed Google Scholar, 11Lieberman AP Pitha PM Shin HS Shin ML Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus.Proc Natl Acad Sci USA. 1989; 86: 6348-6352Crossref PubMed Scopus (706) Google Scholar microglia,12Lee SC Liu W Dickson DW Brosnan CF Berman JW Cytokine production by human fetal microglia and astrocytes: differential induction by lipopolysaccharide and IL-1β.J Immunol. 1993; 150: 2659-2667PubMed Google Scholar, 13Chao CC Hu S Sheng WS Peterson PK Tumor necrosis factor-α production by human fetal microglial cells: regulation by other cytokines.Dev Neurosci. 1995; 17: 97-105Crossref PubMed Scopus (86) Google Scholar and possibly neurons.14Breder CD Tsujimoto M Terano Y Scott DW Saper CB Distribution and characterization of tumor necrosis factor-α-like immunoreactivity in the murine central nervous system.J Comp Neurol. 1993; 337: 543-567Crossref PubMed Scopus (188) Google Scholar Although it is clear that TNF-α is present in the CNS during various insults and that resident neural cells can express TNF-α providing a local source of production for this cytokine, divergent views (see, for example, 15Rothwell NJ Luheshi GN Brain TNF: damage limitation or damaged reputation?.Nature Med. 1996; 2: 746-747Crossref PubMed Scopus (37) Google Scholar, 16Steinman L Some misconceptions about understanding autoimmunity through experiments with knockouts.J Exp Med. 1997; 185: 2039-2041Crossref PubMed Scopus (88) Google Scholar) have evolved as to the role of TNF-α in these pathological states with experimental evidence supporting both detrimental and protective functions. Various manipulations that reduce TNF-α levels, including administration of anti-TNF-α neutralizing antibodies or a soluble TNF type I receptor protein or treatment with the TNF-suppressing drug Rolipram, ameliorate or even prevent experimental autoimmune encephalomyelitis (EAE)17Ruddle NH Bergman CM McGrath KM Lingenheld EG Grunnet ML Padula SJ Clark RB An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.J Exp Med. 1990; 172: 1193-1200Crossref PubMed Scopus (632) Google Scholar, 18Selmaj K Raine CS Cross AH Anti-tumor necrosis factor therapy abrogates autoimmune demyelination.Ann Neurol. 1991; 1991: 694-700Crossref Scopus (409) Google Scholar, 19Klinkert WEF Kojima K Lesslauer W Rinner W Lassmann H Wekerle H TNF-α receptor fusion protein prevents experimental autoimmune encephalomyelitis and demyelination in Lewis rats: an overview.J Neuroimmunol. 1994; 72: 163-168Abstract Full Text Full Text PDF Scopus (115) Google Scholar, 20Sommer N Loschmann PA Northoff GH Weller M Steinbecher A Steinbach JP Lichtenfels R Meyermann R Reitmuller A Fontana A Dichgans J Martin R The antidepressant Rolipram suppresses cytokine production and prevents autoimmune encephalomyelitis.Nature Med. 1995; 1: 244-248Crossref PubMed Scopus (323) Google Scholar or cerebral malaria21Garcia I Miyazaki Y Araki K Araki M Lucas R Grau GE Milon G Belkaid Y Montixi C Lesslauer W Vassalli P Transgenic mice expressing high levels of soluble TNF-R1 fusion protein are protected from lethal septic shock and cerebral malaria, and are highly sensitive to Listeria monocytogenes and Leishmania major infections.Eur J Immunol. 1995; 25: 2401-2407Crossref PubMed Scopus (133) Google Scholar, 22Lucas R Juillard P Decoster E Redard M Burger D Donati Y Giroud C Monso-Hinard C Kesel TD Buurman WA Moore MW Dayer J-M Fiers W Bluethmann H Grau GE Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane-bound TNF in experimental cerebral malaria.Eur J Immunol. 1997; 27: 1719-1725Crossref PubMed Scopus (164) Google Scholar in mice. Intracisternal injection of TNF-α promotes a vigorous inflammatory response with infiltration of the meninges and ventricles with large numbers of leukocytes.23Saukkonen K Sande S Cioffe C Wolpe S Sherry B Cerami A Tuomanen E The role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis.J Exp Med. 1990; 171: 439-448Crossref PubMed Scopus (308) Google Scholar Finally, numerous in vitro studies also support the notion that TNF-α plays a central role in the evolution of neuroinflammation and as well may contribute directly to degenerative CNS disease. In particular, TNF-α is a potent inducer of both cellular adhesion molecule expression by cerebrovascular endothelial cells24Grau GE Lou J TNF in vascular pathology: the importance of platelet-endothelial interactions.Res Immunol. 1993; 144: 355-363Crossref PubMed Scopus (69) Google Scholar and astrocytes25Satoh J-I Kastrukoff LF Kim SU Cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) in cultured human oligodendrocytes and astrocytes.J Neuropathol Exp Neurol. 1991; 50: 215-226Crossref PubMed Scopus (77) Google Scholar and chemokine expression by microglia26McManus CM Brosnan CF Berman JW Cytokine induction of MIP-1α and MIP-1β in human fetal microglia.J Immunol. 1998; 160: 1449-1455PubMed Google Scholar, 27Hayashi M Luo Y Laning J Strieter RM Dorf ME Production and function of monocyte chemoattractant protein-1 and other β-chemokines in murine glial cells.J Neuroimmunol. 1995; 60: 143-150Abstract Full Text PDF PubMed Scopus (217) Google Scholar and astrocytes27Hayashi M Luo Y Laning J Strieter RM Dorf ME Production and function of monocyte chemoattractant protein-1 and other β-chemokines in murine glial cells.J Neuroimmunol. 1995; 60: 143-150Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 28Hurwitz AA Lyman WD Berman JW Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1.J Neuroimmunol. 1995; 57: 193-198Abstract Full Text PDF PubMed Scopus (126) Google Scholar and promotes demyelination and oligodendrocyte injury.29Louis J-C Magal E Takayama S Varon S CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death.Science. 1993; 259: 689-692Crossref PubMed Scopus (454) Google Scholar, 30Selmaj K Raine CS Farooq M Norton WT Brosnan CF Cytokine cytotoxicity against oligodendrocytes: apoptosis induced by lymphotoxin.J Immunol. 1991; 147: 1522-1529PubMed Google Scholar Against this body of evidence for pro-inflammatory and harmful effects of TNF-α, recent studies in gene knockout mice deficient for the TNF genes or their corresponding TNF receptors have provided evidence for an alternative, possible anti-inflammatory or protective function of this cytokine in CNS disease. Mice deficient for TNF-α31Liu J Marino MW Wong G Grail D Dunn A Bettadapura J Slavin AJ Old L Bernard CCA TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination.Nature Med. 1998; 4: 78-83Crossref PubMed Scopus (495) Google Scholar or TNF-α and TNF-β32Frei K Eugster H-P Bopst M Constantinescu CS Lavi E Fontana A Tumor necrosis factor α and lymphotoxin α are not required for the induction of acute experimental autoimmune encephalomyelitis.J Exp Med. 1997; 185: 2177-2182Crossref PubMed Scopus (174) Google Scholar were not only found to be susceptible to the development of EAE but also invariably exhibited a more severe and protracted form of the inflammatory demyelinating disease. In a similar vein, the neuronal injury caused by cerebral ischemia or excitotoxic amino acids was found to be exacerbated in mice deficient for both the p55 and p75 TNF receptors.33Bruce AJ Bolinf W Kindy MS Peschon J Kraemer PJ Carpenter MK Holtsberg FW Mattson MP Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors.Nature Med. 1996; 2: 788-794Crossref PubMed Scopus (842) Google Scholar However, not all studies using knockout mice support a beneficial action of TNF-α in CNS disease; for example, mice deficient for both TNF-α and TNF-β are resistant to cerebral malaria and show a marked reduction in CNS inflammation.34Rudin W Eugster H-P Bordmann G Bonato J Muller M Yamage M Ryffel B Resistance to cerebral malaria in tumor necrosis factor-α/β-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.Am J Pathol. 1997; 150: 257-266PubMed Google Scholar It should be noted, as has recently been outlined,16Steinman L Some misconceptions about understanding autoimmunity through experiments with knockouts.J Exp Med. 1997; 185: 2039-2041Crossref PubMed Scopus (88) Google Scholar that experiments using the current generation of gene knockout mice to understand the role of cytokines such as TNF-α in disease are confounded by a number of issues that warrant caution in interpreting the results. Transgenic mice with CNS-targeted expression of cytokines offer an alternative approach to gene knockout animals for the study of cytokine functions in the intact CNS.35Campbell IL Stalder AK Chiang C-S Bellinger R Heyser CJ Steffensen S Masliah E Powell HC Gold LH Henriksen SJ Siggins GR Transgenic models to assess the neuropathogenic actions of cytokines in the central nervous system.Mol Psychiatry. 1997; 2: 125-129Crossref PubMed Scopus (84) Google Scholar In the case of TNF-α, development of transgenic mice that express either murine TNF-α from its own promoter with expression apparently in neurons36Probert L Akassoglou K Pasparakis M Kontogeorgos G Kollias G Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor α.Proc Natl Acad Sci USA. 1995; 92: 11294-11298Crossref PubMed Scopus (362) Google Scholar or human TNF-α from the GFAP promoter with expression in astrocytes37Akassoglou K Probert L Kontogeorgos G Kollias G Astrocyte-specific but not neuron-specific transmembrane TNF triggers inflammation and degeneration in the central nervous system of transgenic mice.J Immunol. 1997; 158: 438-445PubMed Google Scholar was reported by the same laboratory. In both cases, transgenic animals appear to have had very high TNF-α expression in the brain and exhibited at an early age severe and invariably lethal neurological deficits in association with extensive CNS inflammation and degenerative pathology. In addition to the possible adverse impact of TNF-α on the normal development of the CNS in these transgenic mice, the severe neurological disorder exhibited by these animals necessitated intervening actions to suppress TNF-α levels to be able to breed transgenic mice for further study. In a separate study, Taupin and colleagues reported that transgenic mice with oligodendrocyte-targeted expression of TNF-α driven by the myelin basic protein promoter failed to develop spontaneous disease but developed more severe disease when induced with EAE.38Taupin V Renno T Bourbonniere L Peterson AC Rodriguez M Owens T Increased severity of experimental autoimmune encephalomyelitis, chronic macrophage/microglial reactivity, demyelination in transgenic mice producing tumor necrosis factor-α in the central nervous system.Eur J Immunol. 1997; 27: 905-913Crossref PubMed Scopus (130) Google Scholar Although in this latter report brain levels of transgene encoded TNF-α were shown to be similar to those found in mice with EAE, the targeted expression of this cytokine to oligodendrocytes could be problematic as these cells are not known to produce this cytokine. In addition, oligodendrocytes may be susceptible to nonspecific injury resulting from transgene expression per se.39Turnley AM Morahan G Okano H Bernard O Mikoshiba K Allison J Bartlett PF Miller JFAP Dysmyelination in transgenic mice resulting from expression of class I histocompatibility molecules in oligodendrocytes.Nature. 1991; 353: 566-569Crossref PubMed Scopus (105) Google Scholar With the aim of addressing the issue of the role of TNF-α in the initiation and perpetuation of disease in the adult CNS, we set out here to generate transgenic mice (termed GFAP-TNFα) with expression of this cytokine targeted to a cell (ie, the astrocyte) that is known to produce TNF-α10Chung IY Benveniste EN Tumor necrosis factor-α production by astrocytes.J Immunol. 1990; 144: 2999-3007PubMed Google Scholar, 11Lieberman AP Pitha PM Shin HS Shin ML Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus.Proc Natl Acad Sci USA. 1989; 86: 6348-6352Crossref PubMed Scopus (706) Google Scholar at levels that did not compromise the development and breeding viability of the animal. To this end we describe two independent lines of transgenic mice that exhibit no detectable spontaneous disease until greater than 4 months of age, after which time a progressive and eventually fatal neurological disorder ensues. On further characterization, florid meningoencephalomyelitis and degenerative disease was found in the CNS of symptomatic GFAP-TNFα mice. Interestingly, disease exacerbation with more extensive lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFα mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-α in the CNS is sufficient to induce a chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury. C57BL/6J × SJL F2 mice used for the development of the transgenic lines and Balb/cByJscid/scid (SCID) mice were obtained from Jackson Laboratories (Bar Harbor, ME). For the breeding of GFAP-TNFαscid/scid mice, transgenic mice of the GT-2 line (see below) were mated with Balb/cByJscid/scid mice. GFAP-TNFα × Balb/cByJscid/+ F1 mice were backcrossed with Balb/cByJscid/scid mice, and SCID offspring were identified by analysis of plasma IgG levels using an immunodiffusion assay kit (ICN, Costa Mesa, CA). All mice were maintained in specific-pathogen-free conditions in the closed breeding colony of the Scripps Research Institute. The CNS- and astrocyte-specific expression obtained for fusion gene constructs under the control of the murine GFAP promoter is well documented.35Campbell IL Stalder AK Chiang C-S Bellinger R Heyser CJ Steffensen S Masliah E Powell HC Gold LH Henriksen SJ Siggins GR Transgenic models to assess the neuropathogenic actions of cytokines in the central nervous system.Mol Psychiatry. 1997; 2: 125-129Crossref PubMed Scopus (84) Google Scholar, 40Campbell IL Powell HC Role of cytokines in demyelinating disease studied in transgenic mice.Methods. 1996; 10: 462-477Crossref PubMed Scopus (10) Google Scholar To obtain astrocyte expression of murine TNF-α, a modified strategy was used to that previously used for the generation of the GFAP-IL641Campbell IL Abraham CR Masliah E Kemper P Inglis JD Oldstone MBA Mucke L Neurologic disease induced in transgenic mice by the cerebral overexpression of interleukin 6.Proc Natl Acad Sci USA. 1993; 90: 10061-10065Crossref PubMed Scopus (873) Google Scholar and GFAP-IL342Chiang C-S Powell HC Gold L Samimi A Campbell IL Macrophage/microglial-mediated primary demyelination and motor disease induced by the central nervous system production of interleukin-3 in transgenic mice.J Clin Invest. 1996; 97: 1512-1524Crossref PubMed Scopus (104) Google Scholar transgenic mice. Briefly, a 2.2-kb Sfi-1/Not-1 fragment containing the murine GFAP promoter and an SV-40 small intron was excised from the GFAP-IL3 plasmid construct42Chiang C-S Powell HC Gold L Samimi A Campbell IL Macrophage/microglial-mediated primary demyelination and motor disease induced by the central nervous system production of interleukin-3 in transgenic mice.J Clin Invest. 1996; 97: 1512-1524Crossref PubMed Scopus (104) Google Scholar and cloned into the vector pGEMEX-1 (Promega, Madison, WI) to generate the construct pGF. Next, a 0.65-kb Sma-1/EcoRV fragment containing the human growth hormone (hGH) polyadenylation signal sequence was cut from the plasmid pIC-hGH (kindly provided by Dr. Jan Allison, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) and ligated into the EcoRV site of pGF to generate the construct pGF.GH. Finally, a 1.8-kbEcoN1/EcoRI genomic DNA (a genomic clone containing the entire murine TNF-α and TNF-β gene locus was kindly provided by Dr. Beutler, Southwestern Medical Center, University of Texas) fragment spanning the coding region of murine TNF-α gene was modified by adding Not-1 linkers and was then ligated into the Not-1 site in pGF.GH. After screening for correct orientation, the GFAP-TNFα fusion gene fragment was excised withSfi-1/EcoRI and purified from plasmid DNA before microinjection into fertilized eggs of C57BL/6J × SJL F2 hybrid mice. Transgenic offspring were identified by slot blot analysis of tail DNA using a 32P-labeled random primer hGH DNA fragment as a probe. Transgenic or control mice were killed, and the organs were then removed and immediately snap frozen in liquid nitrogen. Samples were stored at −70°C pending RNA preparation. Total RNA was extracted with TRIZOL reagent (Gibco-BRL, Grand Island, NY) used according to the manufacturer's instructions. The concentration of RNA was determined by ultraviolet spectroscopy at 260 nm. The production and characterization of the multiprobe RPA probe sets used for the detection of cytokine or chemokine gene expression have been described previously.43Hobbs MV Weigle WO Noonan DJ Torbett BE McEvilly RJ Koch RJ Cardenas GJ Ernst DN Patterns of cytokine gene expression by CD4+ T cells from young and old mice.J Immunol. 1992; 150: 3602-3608Google Scholar, 44Asensio VC Campbell IL Chemokine gene expression in the brain of mice with lymphocytic choriomeningitis.J Virol. 1997; 71: 7832-7840Crossref PubMed Google Scholar To distinguish between transgene-encoded TNF-α (tgTNF) mRNA and the endogenously transcribed TNF-α (eTNF) mRNA, RPA probes were constructed that targeted the hGH sequence (to detect tgTNF) (GenBank accession number M13438; nucleotides (nt) 2453 to 2653) or the TNF-α 3′ untranslated region (UTR; to detect eTNF) (GenBank accession number M11731; nt 1110 to 1357). In addition, to detect total TNF-α mRNA, a previously described43Hobbs MV Weigle WO Noonan DJ Torbett BE McEvilly RJ Koch RJ Cardenas GJ Ernst DN Patterns of cytokine gene expression by CD4+ T cells from young and old mice.J Immunol. 1992; 150: 3602-3608Google Scholar RPA probe was used. In all cases, a fragment of the RPL32-4A gene45Dudov KP Perry RP The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene.Cell. 1984; 37: 457-468Abstract Full Text PDF PubMed Scopus (282) Google Scholar served as an internal loading control. RPAs were performed as described previously.46Stalder AK Campbell IL Simultaneous analysis of multiple cytokine receptor mRNAs by RNAse protection assay in LPS-induced endotoxemia.Lymphokine Cytokine Res. 1994; 13: 107-112PubMed Google Scholar For routine histology, organs were removed, fixed in 4% buffered paraformaldehyde, and embedded in paraffin for hematoxylin and eosin or luxol fast blue staining, immunohistochemistry, and in situhybridization. In addition, some fixed brain and spinal cord specimens were transferred to 20% sucrose in PBS and stored overnight at 4°C. After cryoprotection, the tissue was then snap frozen in liquid nitrogen and 10-μm cryostat sections prepared for oil red O staining to visualize neutral fat. Briefly, stock oil red O (Sigma Chemical Co., St. Louis, MO) was prepared at 3 mg/ml in 99% isopropanol. Immediately before use, stock was diluted 1:4 in distilled H2O to obtain a working solution. Sections were stained for 15 minutes in working solution, counterstained for 1 minute in Ehrlichs hematoxylin, rinsed in tap water, coverslipped using an aqueous mounting medium (Aquamount, Lerner Laboratories, Pittsburgh, PA), and immediately inspected by light microscopy. For in situ hybridization, the TNF-α probe used was35S-labeled, single-stranded antisense or sense RNA to murine TNF-α. For probe synthesis, a cDNA for murine TNF-α (kindly provided by Genentech, South San Francisco, CA) was subcloned into pBluescript-KS (Stratagene, La Jolla, CA) and antisense or sense RNAs synthesized using T7 or T3 RNA polymerase, respectively. In situ hybridization was performed as described by Simmons et al47Simmons DM Arriza JL Swanson LW A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes.J Histotechnol. 1989; 12: 169-181Crossref Google Scholar with modifications. Briefly, paraffin sections were deparaffinized and rehydrated in graded alcohols. After post-fixation in 4% formaldehyde, proteinase K treatment (2.4 mg/100 ml of 5X TE buffer at 37°C for 15 minutes) and acetylation (250 μl of acetic anhydride in 100 ml of PBS for 10 minutes), the slides were dehydrated in graded alcohol and dried. The 35S-labeled sense or antisense probes were hybridized to the tissue overnight at 56°C. After digestion with RNAse A (Promega), slides were washed in decreasing concentrations of SSC buffer. After the last SSC step, slides were blocked with serum (Vector Laboratories, Burlingame, CA) and immunohistochemistry was performed where applicable. The primary antibody against GFAP (Dako, Carpinteria, CA) was incubated overnight at 4°C. Antibody-labeled cells were detected using a Vectastain kit (Vector) according to the manufacturer's instructions. After dehydration in graded alcohol, slides were air dried and exposed for 5 days to Cronex film (DuPont, Wilmington, DE). Slides were then dipped in Kodak NTB-2 emulsion, dried, and stored in the dark for 2 weeks. Subsequently, slides were developed, counterstained with Mayer's hematoxylin, and examined by bright field microscopy. Immunohistochemistry for the detection of TNF-α protein was performed on paraformaldehyde-fixed, paraffin-embedded sections. After dewaxing and rehydration, slides were incubated in 1.5% normal goat serum (Vector) in PBS containing 0.1% Saponin (Sigma) for 30 minutes. After blocking, slides were incubated overnight at room temperature with a polyclonal rabbit anti-murine TNF-α antibody (Genzyme, Cambridge, MA) diluted 1:1000 in the blocking buffer. All following steps were performed using the Vectastain kit (Vector) according to the manufacturer's instructions. For immunophenotyping and cellular adhesion molecule immunostaining, mice were killed and organs were removed and immediately snap frozen in isopentane and stored at −70°C until sectioning. Sagittal cryomicrotome cut sections of 10 μm were air dried and either stored dehydrated at −70°C or directly processed. Immediately before staining, tissue sections were fixed in cold (−20°C) acetone/methanol (1:1) for 45 seconds and nonspecific binding was blocked by incubating sections for 30 minutes in PBS containing 3% rabbit and 3% goat serum. Sections were then incubated for 2 hours at room temperature in rat monoclonal antibodies to identify leukocytes (CD45 from Pharmingen, San Diego, CA), lymphocytes (CD4, CD8, and B220 from Pharmingen), NK cells (DX5; Pharmingen), neutrophils (MCA771; Serotec, Raleigh, NC), activation markers (MHC class II, clone M5/114 and Mac-1, and clone TIB 126 from American Type Culture Collection, Rockville, MD), and vascular or cellular adhesion molecules (MAdCAM, VCAM-1, and Endoglin from Pharmingen and ICAM-1 clone YN11.1 kindly provided by Dr. F. Takei, Toronto, Canada). All antibodies were used at a final concentration of 5 μg/ml diluted in the blocking buffer. Bound antibody was detected using a biotinylated anti-rat antibody (Southern Biotechnology Associates, Birmingham, AL) followed by avidin-labeled horseradish peroxidase (Sigma). Staining used 3′,3′-diaminobenzidine (S" @default.
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• W2077896051 title "Late-Onset Chronic Inflammatory Encephalopathy in Immune-Competent and Severe Combined Immune-Deficient (SCID) Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor" @default.
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