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Clinical and Immunopathologic Alterations in Rhesus Macaques Affected with Globoid Cell Leukodystrophy

2007; Elsevier BV; Volume: 172; Issue: 1 Linguagem: Inglês

10.2353/ajpath.2008.070404

1525-2191

Juan T. Borda, Xavier Álvarez, Mahesh Mohan, Marion S. Ratterree, Kathrine Phillippi‐Falkenstein, Andrew A. Lackner, Bruce A. Bunnell,

Alzheimer's disease research and treatments

Globoid cell leukodystrophy, or Krabbe's disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe's disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68+, HAM56+, LN5+, CD163+, IBA-1+, and Glut-5+, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-α, and IL-1β were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP+ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe's disease. Globoid cell leukodystrophy, or Krabbe's disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe's disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68+, HAM56+, LN5+, CD163+, IBA-1+, and Glut-5+, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-α, and IL-1β were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP+ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe's disease. Globoid cell leukodystrophy (GLD; also known as Krabbe's disease) is a rapidly progressing hereditary autosomal recessive neurological disease.1Wenger DA Rafi MA Luzi P Molecular genetics of Krabbe disease (globoid cell leukodystrophy): diagnostic and clinical implications.Hum Mutat. 1997; 10: 268-279Crossref PubMed Scopus (189) Google Scholar, 2Suzuki K Twenty five years of the "psychosine hypothesis": a personal perspective of its history and present status.Neurochem Res. 1998; 23: 251-259Crossref PubMed Scopus (250) Google Scholar, 3Suzuki K Globoid cell leukodystrophy (Krabbe disease): update.J Child Neurol. 2003; 18: 595-603Crossref PubMed Scopus (198) Google Scholar, 4Wenger DA Murine, canine and non-human primate models of Krabbe disease.Mol Med Today. 2000; 6: 449-451Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Wenger DA Sattler M Hiatt W Globoid cell leukodystrophy: deficiency of lactosyl ceramide beta-galactosidase.Proc Natl Acad Sci USA. 1974; 71: 854-857Crossref PubMed Scopus (70) Google Scholar, 6Wenger DA Victoria T Rafi MA Luzi P Vanier MT Vite C Patterson DF Haskins MH Globoid cell leukodystrophy in Cairn and West Highland white terriers.J Hered. 1999; 90: 138-142Crossref PubMed Scopus (56) Google Scholar, 7Wenger DA Suzuki K Suzuki Y Suzuki K Scriver CR Beaudet AL Sly WS Valle D Childs B Kinzler KW Vogelstein B Galactosylceramide lipidosis: globoid cell leukodystrophy (Krabbe disease). The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3669-3687Google Scholar GLD is one of a group of approximately 40 diseases known as lysosomal storage diseases (LSDs) that share common clinical and biochemical properties. The LSDs are a direct consequence of mutation(s) in the gene(s) involved in the generation of enzymes responsible for the turnover or transport of normal cellular metabolic components. As a consequence, the substrate of the defective enzyme accumulates within lysosomes of cells that use it in their metabolic processes and disrupts the normal biological pathway(s). Although they are individually rare, LSDs such as Tay-Sachs disease, Krabbe's disease, various mucopolysaccharidoses, and sphingolipidoses collectively occur in approximately 1 in 5000 live births, with an average life expectancy across the diseases of about 15 years. The incidence of Krabbe's disease, specifically, in the United States is 1 in 100,000 live births. GLD is defined by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC is responsible for lysosomal hydrolysis of several galactolipids, including galactosylceramide, a major sphingolipid of the white matter of the central nervous system, galactosylsphingosine (psychosine), and galactosyldigluceride.4Wenger DA Murine, canine and non-human primate models of Krabbe disease.Mol Med Today. 2000; 6: 449-451Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 8Jesionek-Kupnicka D Majchrowska A Krawczyk J Wendorff J Barcikowska M Lukaszek S Liberski PP Krabbe disease: an ultrastructural study of globoid cells and reactive astrocytes at the brain and optic nerves.Folia Neuropathol. 1997; 35: 155-162PubMed Google Scholar, 9Luzi P Rafi MA Victoria T Baskin GB Wenger DA Characterization of the rhesus monkey galactocerebrosidase (GALC) cDNA and gene and identification of the mutation causing globoid cell leukodystrophy (Krabbe disease) in this primate.Genomics. 1997; 42: 319-324Crossref PubMed Scopus (62) Google Scholar, 10Giri S Jatana M Rattan R Won JS Singh I Singh AK Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease.FASEB J. 2002; 16: 661-672Crossref PubMed Scopus (88) Google Scholar GALC deficiency results in the accumulation of psychosine, which results in apoptotic death of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system, and a markedly shortened life span in humans and animals with this disease.3Suzuki K Globoid cell leukodystrophy (Krabbe disease): update.J Child Neurol. 2003; 18: 595-603Crossref PubMed Scopus (198) Google Scholar, 11Sigurdson CJ Basaraba RJ Mazzaferro EM Gould DH Globoid cell-like leukodystrophy in a domestic longhaired cat.Vet Pathol. 2002; 39: 494-496Crossref PubMed Scopus (27) Google Scholar The clinical manifestations of each lysosomal storage disease depend on the nature of the storage products and the organs affected. The pathogenesis of GLD is usually severe and rapidly progressive. Infants present with pronounced irritability, hypersensitivity to external stimuli, hypotonicity, blindness, and deafness.12D'Agostino AN Sayre GP Hayles AB Krabbe's disease. Globoid cell type of leukodystrophy.Arch Neurol. 1963; 8: 82-96Crossref PubMed Scopus (43) Google Scholar, 13Gullotta F Pavone L Mollica F Grasso S Valenti C Krabbe's disease with unusual clinical and morphological features.Neuropadiatrie. 1979; 10: 395-400Crossref PubMed Scopus (12) Google Scholar, 14Hagberg B Sourander P Svennerholm L Diagnosis of Krabbe's infantile leucodystrophy.J Neurol Neurosurg Psychiatry. 1963; 26: 195-198Crossref PubMed Scopus (50) Google Scholar, 15Suzuki K Suzuki K The twitcher mouse. A model of human globoid cell leukodystrophy (Krabbe's disease).Am J Pathol. 1983; 111: 394-397PubMed Google Scholar Human infants affected by Krabbe's disease show a rapid deterioration in a variety of mental and neuromotor skills. Krabbe-affected infants deteriorate rapidly, and survival beyond two years is uncommon. However, there is phenotypic variability in the age of onset and clinical signs in infants affected with GLD. The histopathological hallmark of this disease is the appearance of globoid cells in the white matter of the central nervous system, located predominantly around blood vessels. Globoid cells are composed of macrophages that have accumulated large amounts of glycolipids in their cytoplasm. In addition to the formation of globoid cells, there is extensive loss of myelin and astrocytosis in the white matter of the central nervous system. In peripheral nerves, axonal degeneration, fibrosis, and macrophage infiltration are often present.16Suzuki K Suzuki Y Galactosylceramide lipidosis: Globoid cell leukodystrophy (Krabbe disease).in: Stanbury JW Wyngaarden JB Fredrickson DS Goldstein JI Brown MS The Metabolic Basis of Inherited Disease. McGraw Hill, New York1983: 857-880Google Scholar Several animal models of Krabbe's disease have been described in species such as the mouse, dog, cat, sheep, and rhesus monkey.4Wenger DA Murine, canine and non-human primate models of Krabbe disease.Mol Med Today. 2000; 6: 449-451Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Wenger DA Victoria T Rafi MA Luzi P Vanier MT Vite C Patterson DF Haskins MH Globoid cell leukodystrophy in Cairn and West Highland white terriers.J Hered. 1999; 90: 138-142Crossref PubMed Scopus (56) Google Scholar, 11Sigurdson CJ Basaraba RJ Mazzaferro EM Gould DH Globoid cell-like leukodystrophy in a domestic longhaired cat.Vet Pathol. 2002; 39: 494-496Crossref PubMed Scopus (27) Google Scholar, 17Suzuki K Genetic galactosylceramidase deficiency (globoid cell leukodystrophy). Krabbe disease in different mammalian species.Neurochem Pathol. 1985; 3: 53-68Crossref PubMed Scopus (35) Google Scholar, 18Kobayashi S Chiu FC Katayama M Sacchi RS Suzuki K Expression of glial fibrillary acidic protein in the CNS and PNS of murine globoid cell leukodystrophy, the twitcher.Am J Pathol. 1986; 125: 227-243PubMed Google Scholar, 19Suzuki K The twitcher mouse: a model for Krabbe disease and for experimental therapies.Brain Pathol. 1995; 5: 249-258Crossref PubMed Scopus (99) Google Scholar, 20Baskin GB Ratterree M Davison BB Falkenstein KP Clarke MR England JD Vanier MT Luzi P Rafi MA Wenger DA Genetic galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys (Macaca mulatta).Lab Anim Sci. 1998; 48: 476-482PubMed Google Scholar, 21Victoria T Rafi MA Wenger DA Cloning of the canine GALC cDNA and identification of the mutation causing globoid cell leukodystrophy in West Highland White and Cairn terriers.Genomics. 1996; 33: 457-462Crossref PubMed Scopus (76) Google Scholar The rhesus monkey model represents the first reported observation of a lysosomal storage disease in any nonhuman primate species. The mutation causing the disease in the rhesus monkey has been identified as a 2-bp deletion at positions 387 and 388 in exon 4 that results in a frameshift and preliminary stop codon.9Luzi P Rafi MA Victoria T Baskin GB Wenger DA Characterization of the rhesus monkey galactocerebrosidase (GALC) cDNA and gene and identification of the mutation causing globoid cell leukodystrophy (Krabbe disease) in this primate.Genomics. 1997; 42: 319-324Crossref PubMed Scopus (62) Google Scholar Newborn macaques homozygous for the gene defect appear normal at birth but decline rapidly. Clinical signs observed in affected animals include moderate to severe muscle tremors of head and limbs, ataxia, and hypermetria. Many of the affected animals develop pronounced tremors and difficulty ambulating, as well as respiratory difficulties including a double inspiratory effort. All of the affected animals have marked decreases in nerve conduction velocities.22Weimer MB Gutierrez A Baskin GB Borda JT Veazey RS Myers L Phillippi-Falkenstein KM Bunnell BA Ratterree MS England JD Serial electrophysiologic studies in rhesus monkeys with Krabbe disease.Muscle Nerve. 2005; 32: 185-190Crossref PubMed Scopus (10) Google Scholar The survival times of affected animals varies from less than one week to as long as 22 months. The focus of our ongoing research in the nonhuman primate model is the development of a link between the observed clinical signs of disease and pathological alterations. Of particular interest is the origin and role, if any, of the globoid cells in disease pathogenesis. 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Perivascular macrophages and parenchymal microglia are the two brain macrophage populations most commonly involved in response to CNS lesions. Perivascular macrophages include blood-derived monocytes that recently immigrated into the brain from the blood. Parenchymal microglia are the resident macrophages of the brain. 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The origin of the globoid cells is defined, and it was demonstrated that activation of microglia/macrophages play a role in the progression of Krabbe's disease in the nonhuman primate model. All animals with Krabbe's disease and the controls were Indian-origin rhesus macaques (Macaca mulatta). Two of the affected macaques were males (EJ72, DH31) and one was female (EA75). Infants were kept with their mothers in appropriate caging equipped with various enrichment devices until disease progression warranted euthanasia. A commercial nonhuman primate biscuit was provided twice daily. Water was available ad libitum. Supplemental fruits and forage were provided throughout the week. The Tulane National Primate Research Center's (TNPRC) Institutional Animal Care and Use Committee approved all studies and procedures. Animal weights were obtained, and a veterinarian performed a complete physical examination monthly. Nerve conduction studies and structural magnetic resonance images (MRIs) were collected monthly on all affected animals.22Weimer MB Gutierrez A Baskin GB Borda JT Veazey RS Myers L Phillippi-Falkenstein KM Bunnell BA Ratterree MS England JD Serial electrophysiologic studies in rhesus monkeys with Krabbe disease.Muscle Nerve. 2005; 32: 185-190Crossref PubMed Scopus (10) Google Scholar Motor maturity items on neurobehavioral assessment scales standardized for use with infant rhesus macaques37Champoux M DeGregorio G Schneider ML Suomi SJ Inanimate environmental enrichment for group-housed rhesus macaque infants.Am J Primatol. 1990; 22: 61-67Crossref Scopus (11) Google Scholar, 38Schneider ML Suomi SJ The effect of mild stress during pregnancy on birthweight and neuromotor maturation in rhesus monkey infants (M. mulatta).Infant Behav Dev. 1992; 15: 389-403Crossref Scopus (93) Google Scholar, 39Schneider ML Suomi SJ Neurobehavioral assessment in rhesus monkey neonates (Macaca mulatta): developmental changes, behavioral stability, and early experience.Infant Behav Dev. 1992; 15: 155-177Crossref Scopus (117) Google Scholar were used to assess affected infants through 10 months of age, provided their health status permitted testing. Scores were compared with scores obtained from 24 unaffected, normal rhesus infants at the TNPRC. Animals affected with Krabbe's disease were euthanized on the advice of the attending veterinarian once the disease progressed to the point that the animal had difficulty swallowing/eating, significant and consistent weight loss, and/or respiratory difficulties. Macaques were humanely euthanized with an i.v. overdose of pentobarbital. Age-matched tissues from three unaffected macaques of 2, 20, and 27 months of age were examined in parallel. A complete necropsy was performed, and all tissues were collected immediately after euthanasia. Tissues were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. Selected neural tissues were also stained with periodic acid-Schiff, Luxol Fast blue/PAS, Alcian Blue (pH 2.5) and Safranin O. Tissues adjacent to those fixed in formalin were embedded in optimal cutting temperature compound (O.C.T.; Miles Inc., Elkhart, IN), snap frozen by immersion in 2-methylbutane in dry ice, and cut into sections 6 to 8 μm thick for immunohistochemistry and confocal microscopy, and at 100 μm for RNA extraction for PCR. To evaluate neural lesions in affected animals and define the immunophenotype of cells involved, we used immunohistochemistry and multilabel confocal microscopy using specific cell markers. Single-label immunohistochemistry for myelin-producing cells (anti-CNPase, IgG1; Sigma, Saint Louis, MO), microglia (anti-IBA 1, polyclonal; Wako Pure Chemical Industries, Richmond, VA), astrocytes (anti-glial fibrillary acidic protein, IgG1; Sigma; and anti-peripherin, IgG2b; Novocastra Laboratories Ltd., Newcastle, UK), and neurons (anti-neuronal nuclei, IgG1, Chemicon, Temecula, CA) was performed as previously described by Borda et al40Borda JT Alvarez X Kondova I Aye P Simon MA Desrosiers RC Lackner AA Cell tropism of simian immunodeficiency virus in culture is not predictive of in vivo tropism or pathogenesis.Am J Pathol. 2004; 165: 2111-2122Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Tissue sections were incubated sequentially with the primary, cell-type-specific antibody for 60 minutes (monoclonal) or 30 minutes (polyclonal) at room temperature followed by biotinylated horse anti-mouse or goat anti-rabbit (Vector Laboratories, Burlingame, CA) secondary antibodies, respectively. Finally, sections were incubated with avidin-biotin complex for 30 minutes, and the reaction was visualized with 3,3′-diaminobenzidine (DAB; Dako, Carpinteria, CA) as the chromogen.41Babcock AM Standing D Bullshields K Schwartz E Paden CM Poulsen DJ In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference.Mol Ther. 2005; 11: 899-905Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar As a negative control, serial sections were processed identically using equivalent concentrations of irrelevant primary antibodies of the same isotype or preimmmune rabbit serum. To evaluate more rigorously the immunophenotype of cells involved in neural lesions of affected animals, multilabel immunofluorescence and confocal microscopic analyses on formalin-fixed tissues and tissue samples embedded in OCT were performed as described previously by Williams and colleagues.36Williams KC Corey S Westmoreland SV Pauley D Knight H deBakker C Alvarez X Lackner AA Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.J Exp Med. 2001; 193: 905-915Crossref PubMed Scopus (332) Google Scholar Tissues were cut into sections 6 to 8 μm thick and fixed in 4% paraformaldehyde for 20 minutes. The monoclonal and polyclonal antibodies used for this analysis are listed in Table 1.Table 1Antibodies Used in Immunohistochemistry and ImmunofluorescenceAntigenCell typeSourceAntibody type*For monoclonal antibodies the isotype is indicated. All polyclonal antibodies were made in rabbit.Dilution workingIFAIHCCD68MacrophageDakoIgG1, kappa1:201:125HAM56MacrophageDakoIgM1:101:25LN5MacrophageZymed (South San Francisco, CA)IgM1:101:200CD163MacrophageNovocastraIgG11:501:100MAC 387Mono/macrophageDakoIgG1, kappa1:201:100MRP8Mono/macrophageAccurate Chemical & Scientific Corp.IgG11:201:100IBA-1Resting microgliaBD PharMingen (San Diego, CA)IgG1,Kappa1:100HLA-DRActivationZymedIgG2b1:201:50Glut-5MicrogliaChemiconPolyclonal1:500CD11bMicroglia/macrophageNovocastraIgG11:10Neu-NNeuronChemiconIgG11:101:200GFAPAstrocyteSigmaIgG1, labeled with CY31:3001:3000PeripherinAstrocyteNovocastraIgG2b1:201:100S-100Glial, ependymal, Schwann cellsSigmaPolyclonal1:100CNPaseOligodendrocyteSigmaIgG1Schwann cells1:100Glut-1Endothelial cellsChemiconPolyclonal1:20iNOS/NOS Type IIBD PharMingenIgG2a1:251:125CCL2 (MCP-1)AbcamPolyclonal1:201:100* For monoclonal antibodies the isotype is indicated. All polyclonal antibodies were made in rabbit. Open table in a new tab For immunohistochemistry, brain sections were washed for 15 minutes in phosphate-buffered saline containing 0.2% fish skin gelatin (PBS-FSG), blocked with Dako protein block, and incubated with the primary antibody for 1 hour at room temperature. Sections were washed for 15 minutes with PBS-FSG incubated with the fluorescently labeled secondary antibody for 30 minutes at room temperature. Secondary antibodies were diluted in Dako antibody diluent at 1:1000 and included anti-mouse or anti-rabbit Alexa 488 (green), Alexa 568 (red), and Alexa 633 (far red) (all from Invitrogen, Carlsbad, CA). After antibody treatment, sections were washed twice for 15 minutes in PBS-FSG. Finally, the sections were rinsed in double distilled water (ddH2O) and mounted on coverslips with aqueous mounting medium. If a second primary antibody and/or a third primary antibody was used (double-label and triple-label) they were applied sequentially after the first primary antibody, and both or all were developed simultaneously. Some sections were stained with Topro-3 (Invitrogen) to delineate the nuclei of cells by incubation for 5 minutes at 1 μg/ml followed by a PBS wash. To identify the inducible nitric oxide synthase (iNOS)- and CCL2-expressing cells, a complete examination of affected and normal control brain was performed using immunohistochemistry and multilabeled confocal microscopy. To define the immunophenotype of CCL2-producing cells, we used a rabbit polyclonal to CCL2 (Abcam Inc., Cambridge, MA) combined with GFAP (anti-glial fibrillary acidic protein, IgG1 conjugated to Cy3; Sigma) and Peripherin (IgG2b, Novocastra Laboratories), both markers for intermediate filaments expressed by astrocytes in the CNS of macaques.42Mathew JS Westmoreland S Alvarez X Simon MA Pauley DR MacKey JJ Lackner AA Expression of peripherin in the brain of macaques (Macaca mulatta and M. fascicularis) occurs in astrocytes rather than neurons and is associated with encephalitis.Neuropathol Appl Neurobiol. 2001; 27: 434-443Crossref PubMed Scopus (6) Google Scholar To identify the iNOS-expressing cells, we used iNOS/NOS type II monoclonal antibody (IgG2a; BD Biosciences, San Jose, CA) combined with the following markers for microglia/macrophage CD11b (IgG1; Novocastra Laboratories) and CD68 (IgG1; Dako Corporation), neurons NeuN (anti-neuronal nuclei, IgG1; Chemicon), astrocytes (anti-glial fibrillary acidic protein, IgG1, conjugated to Cy3; Sigma), and endothelial cells Glut1 (polyclonal; Chemicon). To evaluate and quantify the presence of iNOS, brain sections of affected animals and matched tissues from unaffected macaques were stained for iNOS using immunohistochemistry (iNOS/NOS type II, IgG2a; BD Biosciences). Sections were incubated with the primary antibody for 1 hour at room temperature, followed by biotinylated anti-mouse secondary antibodies43Friedenstein AJ Deriglasova UF Kulagina NN Panasuk AF Rudakowa SF Luria EA Ruadkow IA Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.Exp Hematol. 1974; 2: 83-92PubMed Google Scholar for 30 minutes. Finally, sections were incubated with avidin-biotin complex for 30 minutes, and the reaction was visualized with DAB43Friedenstein AJ Deriglasova UF Kulagina NN Panasuk AF Rudakowa SF Luria EA Ruadkow IA Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.Exp Hematol. 1974; 2: 83-92PubMed Google Scholar as the chromogen. For quantification of iNOS expression, images of ten randomly selected areas of 0.5 mm2 were collected for each slide from each animal; five areas from gray matter and five from white matter. The images were collected using a Leica DM50 microscope with a SPOT Insight digital camera (Diagnostics Instruments Inc., Sterling Heights, MI). The light, exposure time, and magnification were kept constant to allow comparison among the tissues. Image analysis of the tissues was performed by measuring area and intensity of labeling for iNOS in the images with the NIH Image software (version 1.63), as we have described previously.44Williams K Schwartz A Corey S Orandle M Kennedy W Thompson B Alvarez X Brown C Gartner S Lackner A Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.Am J Pathol. 2002; 161: 575-585Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipp