Excitotoxic Brain Injury Stimulates Expression of the Chemokine Receptor CCR5 in Neonatal Rats
1998; Elsevier BV; Volume: 153; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65752-5
ISSN1525-2191
AutoresJohn M. Galasso, Jeffrey K. Harrison, Faye S. Silverstein,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoChemokines interact with specific G-protein-coupled receptors to activate and direct recruitment of immune cells. Some chemokines are up-regulated in pathological conditions of the central nervous system, and recently several chemokine receptors, including CCR5, were identified in the brain. However, little is known about the regulation of expression of chemokine receptors in the brain. Direct intracerebral injection of N-methyl-d-aspartate (NMDA), an excitatory amino acid agonist, elicits reproducible focal excitotoxic brain injury; in neonatal rats, intrahippocampal NMDA injection stimulates expression of pro-inflammatory cytokines and elicits a robust microglia/monocyte response. We hypothesized that NMDA-induced neurotoxicity would also stimulate expression of CCR5 in the neonatal rat brain. We evaluated the impact of intrahippocampal injections of NMDA on CCR5 expression in postnatal day 7 rats. Reverse transcription polymerase chain reaction revealed an increase in hippocampal CCR5 mRNA expression 24 hours after lesioning, and in situhybridization analysis demonstrated that CCR5 mRNA was expressed in the lesioned hippocampus and adjacent regions. Western blot analysis demonstrated increased CCR5 protein in hippocampal tissue extracts 32 hours after lesioning. Complementary immunocytochemistry studies identified both infiltrating microglia/monocytes and injured neurons as the principal CCR5-immunoreactive cells. These results provide the first evidence that acute excitotoxic injury regulates CCR5 expression in the developing rat brain. Chemokines interact with specific G-protein-coupled receptors to activate and direct recruitment of immune cells. Some chemokines are up-regulated in pathological conditions of the central nervous system, and recently several chemokine receptors, including CCR5, were identified in the brain. However, little is known about the regulation of expression of chemokine receptors in the brain. Direct intracerebral injection of N-methyl-d-aspartate (NMDA), an excitatory amino acid agonist, elicits reproducible focal excitotoxic brain injury; in neonatal rats, intrahippocampal NMDA injection stimulates expression of pro-inflammatory cytokines and elicits a robust microglia/monocyte response. We hypothesized that NMDA-induced neurotoxicity would also stimulate expression of CCR5 in the neonatal rat brain. We evaluated the impact of intrahippocampal injections of NMDA on CCR5 expression in postnatal day 7 rats. Reverse transcription polymerase chain reaction revealed an increase in hippocampal CCR5 mRNA expression 24 hours after lesioning, and in situhybridization analysis demonstrated that CCR5 mRNA was expressed in the lesioned hippocampus and adjacent regions. Western blot analysis demonstrated increased CCR5 protein in hippocampal tissue extracts 32 hours after lesioning. Complementary immunocytochemistry studies identified both infiltrating microglia/monocytes and injured neurons as the principal CCR5-immunoreactive cells. These results provide the first evidence that acute excitotoxic injury regulates CCR5 expression in the developing rat brain. Recent experimental data indicate that inflammatory mediators contribute substantially to the pathogenesis of neonatal brain injury.1Silverstein FS Barks JDE Hagan P Liu XH Ivacko J Szaflarski J Cytokines and perinatal brain injury.Neurochem Int. 1997; 30: 375-383Crossref PubMed Scopus (147) Google Scholar Heightened interest in the pathogenetic role of inflammatory mediators in the immature nervous system stems from clinical observations linking detection of pro-inflammatory cytokines in the amniotic fluid and in the neonatal brain with adverse neurodevelopmental outcome.2Dammann O Leviton A Maternal intrauterine infection, cytokines, and brain damage in the preterm newborn.Pediatr Res. 1997; 42: 1-8Crossref PubMed Scopus (809) Google Scholar, 3Yoon BH Jun JK Romero R Park KH Gomez R Choi JH Kim IO Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1β, and tumor necrosis factor-α), neonatal brain white matter lesions, and cerebral palsy.Am J Obstet Gynecol. 1997; 177: 19-26Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar Studies in experimental models of hypoxic-ischemic and excitotoxic neonatal brain injury provide direct evidence for associated activation of pro-inflammatory mechanisms.4Szaflarski J Burtrum D Silverstein FS Cerebral hypoxia-ischemia stimulates cytokine gene expression in perinatal rats.Stroke. 1995; 26: 1093-1100Crossref PubMed Scopus (261) Google Scholar, 5Ivacko JA Sun R Silverstein FS Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats.Pediatr Res. 1996; 39: 1-9Crossref PubMed Scopus (135) Google Scholar, 6Hagan P Barks JDE Yabut M Davidson BL Roessler B Silverstein FS Adenovirus-mediated over-expression of interleukin-1 receptor antagonist reduces susceptibility to excitotoxic brain injury in perinatal rats.Neuroscience. 1996; 75: 1033-1045Crossref PubMed Scopus (60) Google Scholar, 7Szaflarski J Ivacko J Liu XH Warren JS Silverstein FS Excitotoxic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.Mol Brain Res. 1998; 55: 306-314Crossref PubMed Scopus (33) Google Scholar, 8Ivacko J Szaflarski J Malinak C Flory C Warren JS Silverstein FS Hypoxic-ischemic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.J Cereb Blood Flow Metab. 1997; 17: 759-770Crossref PubMed Scopus (95) Google Scholar, 9Hagan P Poole S Bristow AF Tilders F Silverstein FS Intracerebral NMDA injection stimulates production of interleukin-1β in perinatal rat brain.J Neurochem. 1996; 67: 2215-2218Crossref PubMed Scopus (54) Google Scholar The maturational stage of postnatal day (P)7 rat brain corresponds roughly with late-gestation human brain maturation. A well characterized model of acute excitotoxic brain injury, elicited by direct intracerebral (i.c.) administration of the glutamate agonist N-methyl-d-aspartate (NMDA) into P7 rat brain10McDonald JW Silverstein FS Johnston MV Neurotoxicity of N-methyl-d-aspartate is markedly enhanced in developing rat central nervous system.Brain Res. 1988; 459: 200-203Crossref PubMed Scopus (443) Google Scholar facilitates analysis of the molecular and cellular responses elicited by NMDA receptor over-activation in vivo. In the rat, NMDA-induced brain injury is dose dependent and reproducible; susceptibility to NMDA-induced neurotoxicity peaks at P7.10McDonald JW Silverstein FS Johnston MV Neurotoxicity of N-methyl-d-aspartate is markedly enhanced in developing rat central nervous system.Brain Res. 1988; 459: 200-203Crossref PubMed Scopus (443) Google Scholar Over-activation of the NMDA-subtype glutamate receptor leads to increased intracellular calcium accumulation and increased neuronal nitric oxide production; a complex cascade of downstream molecular mechanisms, including generation of soluble injury mediators, determine the ultimate extent of tissue damage.11Choi DW Calcium: still center-stage in hypoxic-ischemic neuronal death.Trends Neurosci. 1995; 18: 58-60Abstract Full Text PDF PubMed Scopus (879) Google Scholar Direct i.c. administration of NMDA into P7 rat brain elicits rapid stimulation of interleukin (IL)-1β production,9Hagan P Poole S Bristow AF Tilders F Silverstein FS Intracerebral NMDA injection stimulates production of interleukin-1β in perinatal rat brain.J Neurochem. 1996; 67: 2215-2218Crossref PubMed Scopus (54) Google Scholar and pharmacological antagonism of IL-1 markedly attenuates injury,6Hagan P Barks JDE Yabut M Davidson BL Roessler B Silverstein FS Adenovirus-mediated over-expression of interleukin-1 receptor antagonist reduces susceptibility to excitotoxic brain injury in perinatal rats.Neuroscience. 1996; 75: 1033-1045Crossref PubMed Scopus (60) Google Scholar indicating the potential of inflammatory cytokines to exacerbate damage. Recent observations suggest that chemokines, a family of small proteins involved in the activation and directed migration of leukocytes,12Premack BA Schall TJ Chemokine receptors: gateways to inflammation and infection.Nature Med. 1996; 2: 1174-1178Crossref PubMed Scopus (573) Google Scholar may be potential mediators of brain injury. Currently, four distinct subfamilies of chemokines have been identified; CC, CXC, C, and CX3C subfamilies are distinguished by the positioning of conserved cysteine residues. These structural distinctions correlate with functional differences in target specificity. For example, CC chemokines primarily recruit and activate monocytes and lymphocytes,13Schall TJ Bacon K Camp RDR Kaspari JW Goeddel DV Human macrophage inflammatory protein-α (MIP-1α) and MIP-1β chemokines attract distinct populations of lymphocytes.J Exp Med. 1993; 177: 1821-1826Crossref PubMed Scopus (500) Google Scholar, 14Carr MW Roth SJ Luther E Rose SS Springer TA Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant.Proc Natl Acad Sci USA. 1994; 91: 3652-3656Crossref PubMed Scopus (1075) Google Scholar, 15Bell MD Taub DD Perry VH Overriding the brain's intrinsic resistance to leukocyte recruitment with intraparenchymal injections of recombinant chemokines.Neuroscience. 1996; 74: 283-292Crossref PubMed Scopus (157) Google Scholar CXC chemokines recruit neutrophils,16Baggiolini M Walz A Kunkel SL Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.J Clin Invest. 1989; 84: 1045-1049Crossref PubMed Scopus (1744) Google Scholar, 17Yoshimura T Matsushima K Tanaka S Robinson EA Appella E Oppenheim JJ Leonard EJ Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.Proc Natl Acad Sci USA. 1987; 84: 9233-9237Crossref PubMed Scopus (917) Google Scholar, 18Walz A Burgener R Car B Baggiolini M Kunkel SL Strieter RM Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin-8.J Exp Med. 1991; 174: 1355-1362Crossref PubMed Scopus (313) Google Scholar lymphotactin (C subfamily) has chemotactic activity for lymphocytes,19Kelner GS Kennedy J Bacon KB Kleyensteuber S Largaespada DA Jenkins NA Copeland NG Bazan JF Moore KW Schall TJ Zlotnik A Lymphotactin: a cytokine that represents a new class of chemokine.Science. 1994; 266: 1395-1399Crossref PubMed Scopus (637) Google Scholar and the newly described CX3C chemokine is chemotactic for lymphocytes and monocytes.20Bazan JF Bacon KB Hardiman G Wang W Soo K Rossi D Greaves DR Zlotnik A Schall TJ A new class of membrane-bound chemokine with a CX3C motif.Nature. 1997; 385: 640-644Crossref PubMed Scopus (1746) Google Scholar Most chemokines elicit their effects through interactions with seven-transmembrane-domain, G-protein-coupled receptors. There has been rapid progress in characterization of multiple distinct human and rodent chemokine receptors.12Premack BA Schall TJ Chemokine receptors: gateways to inflammation and infection.Nature Med. 1996; 2: 1174-1178Crossref PubMed Scopus (573) Google Scholar, 21Dunstan CN Salafranca MN Adhikari S Xia Y Feng L Harrison JK Identification of two rat genes orthologous to the human interleukin-8 receptors.J Biol Chem. 1996; 271: 32770-32776Crossref PubMed Scopus (69) Google Scholar, 22Jiang Y Salafranca MN Adhikari S Xia Y Feng L Sonntag MK deFiebre CM Pennell NA Streit WJ Harrison JK Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis.J Neuroimmunol. 1998; 86: 1-12Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Interest in the potential pathophysiological role of chemokines and their receptors in the developing central nervous system (CNS) stems from several recent findings. First, excitotoxic injury in neonatal rats is characterized by an increase in activated microglia (macrophage-like cells in the CNS) in injured areas;5Ivacko JA Sun R Silverstein FS Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats.Pediatr Res. 1996; 39: 1-9Crossref PubMed Scopus (135) Google Scholar these activated microglia may contribute to the progression of neuronal injury through the release of inflammatory mediators.23Giulian D Vaca K Noonan CA Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1.Science. 1990; 250: 1593-1596Crossref PubMed Scopus (568) Google Scholar, 24Giulian D Wendt E Vaca K Noonan CA The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes.Proc Natl Acad Sci USA. 1993; 90: 2769-2773Crossref PubMed Scopus (204) Google Scholar In addition, acute excitotoxic injury in neonatal rats stimulates gene and protein expression of the chemokine monocyte chemoattractant protein (MCP)-1, a potent regulator of monocytes, in areas where activated microglia/monocytes subsequently accumulate.7Szaflarski J Ivacko J Liu XH Warren JS Silverstein FS Excitotoxic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.Mol Brain Res. 1998; 55: 306-314Crossref PubMed Scopus (33) Google Scholar, 8Ivacko J Szaflarski J Malinak C Flory C Warren JS Silverstein FS Hypoxic-ischemic injury induces monocyte chemoattractant protein-1 expression in neonatal rat brain.J Cereb Blood Flow Metab. 1997; 17: 759-770Crossref PubMed Scopus (95) Google Scholar Chemokine receptors, including CCR5, have been implicated in the pathogenesis of HIV-1 infection.25Alkhatib G Combadiere C Broder CC Feng Y Kennedy PE Murphy PM Berger EA CC CKR5: a RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1.Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2465) Google Scholar, 26Dragic T Litwin V Allaway GP Martin SR Huang Y Nagashima KA Cayanan C Maddon PJ Koup RA Moore JP Paxton WA HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.Nature. 1996; 381: 667-673Crossref PubMed Scopus (2841) Google Scholar Recent evidence also suggests that HIV-1 infection of microglia, the major target cells of HIV-1 in the brain, is in part mediated by CCR527He J Chen Y Farzan M Choe H Ohagen A Gartner S Busciglio J Yang X Hofmann W Newman W Mackay CR Sodroski J Gabuzda D CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia.Nature. 1997; 385: 645-649Crossref PubMed Scopus (825) Google Scholar and that HIV-1-infected microglia secrete neurotoxic factors that may contribute to neuronal death.23Giulian D Vaca K Noonan CA Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1.Science. 1990; 250: 1593-1596Crossref PubMed Scopus (568) Google Scholar, 24Giulian D Wendt E Vaca K Noonan CA The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes.Proc Natl Acad Sci USA. 1993; 90: 2769-2773Crossref PubMed Scopus (204) Google Scholar In experimental models of HIV-1-associated CNS injury, the HIV-1 envelope glycoprotein 120 (gp120) influences susceptibility to NMDA neurotoxicity in P7 hippocampus,28Barks JDE Liu XH Sun R Silverstein FS gp120, a human immunodeficiency virus-1 coat protein, augments excitotoxic hippocampal injury in perinatal rats.Neuroscience. 1997; 76: 397-409Crossref PubMed Scopus (55) Google Scholar and microglia have been implicated as mediators of gp120 neurotoxicity.24Giulian D Wendt E Vaca K Noonan CA The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes.Proc Natl Acad Sci USA. 1993; 90: 2769-2773Crossref PubMed Scopus (204) Google Scholar, 29Lipton SA Requirements for macrophages in neuronal injury induced by HIV envelope protein gp120.NeuroReport. 1992; 3: 913-915Crossref PubMed Scopus (137) Google Scholar Together, these observations prompted us to evaluate whether acute NMDA-mediated brain injury regulates CCR5 expression in the neonatal rat brain. P7 Sprague-Dawley rats were obtained from Charles River (Wilmington, MA). The following reagents were purchased: NMDA (Sigma Chemical Co., St. Louis, MO); Tri-Reagent (Molecular Research Center, Cincinnati, OH); MuLV RT, random hexamers, dNTPs, and RNAse inhibitor (Perkin Elmer, Foster City, CA); DNAse and in vitrotranscription kit (Ambion, Austin TX); restriction endonucleases (Boehringer Mannheim, Indianapolis, IN); and [35S]UTP (NEN Dupont, Boston, MA). Protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate) and teleostean (cold-water fish skin) gelatin were from Sigma. BCA protein assay kit and enhanced luminol were obtained from Pierce (Rockford, IL). The following antibodies and related reagents were also purchased: goat anti-CCR5 antibody (Ab), CCR5 blocking peptide, normal goat IgG, and horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA); ED-1 monoclonal antibody (MAb; Serotec, Oxford, UK); glial fibrillary acidic protein (GFAP) polyclonal Ab (Dako, Carpinteria, CA); all biotinylated secondary Abs, ABC Elite kit, normal sera, and normal mouse and rabbit IgG (Vector Laboratories, Burlingame, CA); stable diaminobenzidine (DAB; Research Genetics, Huntsville, AL). All surgical protocols were approved by the University of Michigan Committee on Care and Use of Animals. All lesioning was performed in P7 Sprague-Dawley rats of both genders, using previously reported methods.30McDonald JW Roeser NF Silverstein FS Johnston MV Quantitative assessment of neuroprotection against NMDA-induced brain injury.Exp Neurol. 1989; 106: 289-296Crossref PubMed Scopus (100) Google Scholar Animals were deeply anesthetized by methoxyfluorane inhalation and placed in a standardized holder; the scalp was incised, and skull surface landmarks were identified. At the injection site, the skull was penetrated with a 22-gauge needle, and a 1-μl Hamilton syringe attached to a 25-gauge needle was used to deliver the NMDA-containing solution (10 nmol of NMDA/0.5 μl) over 2 minutes; stereotaxic coordinates were targeted to the right dorsolateral hippocampus (relative to Bregma: antero-posterior, 2.0 mm; lateral, 2.5 mm; dorsal, 4.0 mm). Thirty minutes later, after full recovery from anesthesia, animals were returned to their dams. Animals were housed in a temperature-regulated incubator (maintained at 37°C) during the recovery period. All experiments included controls that underwent the same procedures, in which an equal volume of PBS was substituted for NMDA, as well as unlesioned littermate controls. Animals were killed either by decapitation or by administration of a lethal dosage of chloral hydrate (3 g/kg) followed by perfusion-fixation. RNA samples were prepared from the left and right hippocampus of animals that received right intrahippocampal injections of 10 nmol of NMDA 8, 16, 24, 48, or 72 hours earlier and from animals that had received PBS injections (24 hours earlier). Normal P8 hippocampus was also collected. Brains were divided along the midline, and left and right hippocampus were microdissected on ice. Four hippocampi were pooled per sample. Three independent samples were prepared from normal P8 rats and from animals that received NMDA injections and were killed 24 hours later. Total RNA was isolated using Tri-Reagent (1.2 ml) according to the manufacturer's directions and stored at −70°C. Concentration and purity of RNA samples were estimated by spectrophotometric analysis. RNA samples were pretreated with DNAse; 1 μg of total RNA was suspended in 10 μl of diethylpyrocarbonate-treated H2O containing 2 U of DNAse in 20 mmol/L Tris/HCl (pH 8.4), 50 mmol/L KCl, and 20 mmol/L MgCl2 (15 minutes at room temperature); the reaction was stopped by the addition of EDTA (final concentration, 2.5 mmol/L), and DNAse was inactivated by heating (65°C for 15 minutes). RT was performed as previously described4Szaflarski J Burtrum D Silverstein FS Cerebral hypoxia-ischemia stimulates cytokine gene expression in perinatal rats.Stroke. 1995; 26: 1093-1100Crossref PubMed Scopus (261) Google Scholar with minor modifications. Briefly, 1 μg of DNAse-treated RNA was incubated with 50 U of MuLV reverse transcriptase, 2 μmol/L random hexamers, 20 U of RNAse inhibitor, and 0.5 mmol/L of each dNTP under the following conditions: 10 minutes at room temperature, 15 minutes at 42°C, and 5 minutes at 99°C. The RT product was diluted to a final volume of 100 μl in sterile H2O. Two sets of oligonucleotide primers were used to co-amplify the RT product: 1) sense (5′-CACCCTGTTTCGCTGTAGGAATG-3′)and antisense (5′-GCAGTGTGTCATCCCAAGAGTCTC-3′) primers to amplify a 219-bp fragment of the rat CCR5 cDNA sequence and 2) primers (5′-TCCTGCACCACCAACTGCTTAG-3′ and 5′-CAGATCCACAACGGATACATTGG-3′) to amplify a 298-bp fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a ubiquitously expressed gene that was used to normalize CCR5 mRNA. In preliminary experiments, optimal MgCl2concentration (1.0 mmol/L), pH 9.0, and primer concentrations (0.2 μmol/L for CCR5 primers and 0.05 μmol/L GAPDH primers) were determined; amplification conditions to yield results within the linear range of amplification for CCR5 and GAPDH were established, using 20 μl of RT product (94°C for 90 seconds, 56°C for 40 seconds, and 72°C for 40 seconds for 33 cycles). All RT-PCR reaction products were visualized in ethidium-bromide-stained 2% agarose gels. Results were quantified by fluorometric scanning of the gels, and measurement of arbitrary optical density units (expressed in counts/mm) of each band was performed using the Molecular Analyst imaging system (Bio-Rad, Hercules, CA); values for CCR5 mRNA in each sample were normalized, based on GAPDH mRNA content/sample. In three independent RT-PCR assays, CCR5 expression was compared in samples from normal P8 samples and in left and right hippocampal samples from animals that had been lesioned 24 hours earlier; values for left and right hippocampal CCR5 mRNA expression were expressed as a percentage of corresponding normal control values and were compared using the Mann-Whitney ranking test. To evaluate the efficacy of the DNAse treatment, a right hippocampal RNA sample (24 hours after NMDA injection) was DNAse treated and amplified using conditions described above, but without RT; no GAPDH or CCR5 fragments were amplified (data not shown). A 310-bp EcoRV/BglII fragment of the rat CCR5 cDNA sequence (GenBank accession U77350), cloned into a pGEM7(+) vector, was used as a template for in vitro transcription reactions to generate 35S-labeled sense and antisense riboprobes. Briefly, the plasmid construct was linearized with eitherHindIII or EcoRI for sense and antisense (c)RNA probes, respectively. [35S]UTP (specific activity, 1100 to 1400 Ci/mmol) was incorporated using an in vitrotranscription kit according to manufacturer's directions. Based on results of RT-PCR assays that showed increased CCR5 mRNA expression at 24 hours after right intrahippocampal NMDA injection, this time point was selected for in situ hybridization analysis. Samples were prepared from animals that had received right intrahippocampal NMDA (10 nmol) injections (n = 5) or PBS (n = 2) and from unlesioned P8 animals (n = 2); preliminary experiments were conducted with two samples from each group to ensure that methods were appropriate for detection of CCR5 mRNA. Using assay conditions that enabled detection of CCR5 mRNA, three additional NMDA-injected brains were assayed; 14 sections/brain were hybridized with the antisense riboprobe, and 6 sections/brain were hybridized with the sense riboprobe. Brains were removed rapidly and frozen in crushed dry ice. Frozen, 20-μm coronal sections were collected on poly-l-lysine-coated slides. Sections were fixed in 4% formaldehyde for 1 hour and washed in PBS. Sections were then treated with proteinase K (5 μg/ml) for 5 minutes at 37°C and acetylated in 0.25% acetic anhydride with rapid stirring for 10 minutes at room temperature. After a 5-minute wash in 2X SSC, sections were dehydrated in graded ethanols. Sections were incubated with riboprobes (106 cpm/slide) in 50% formamide, 10% dextran sulfate, 1 mmol/L EDTA, 10 mmol/L Tris, and 0.1 mmol/L dithiothreitol for 20 hours at 55°C. On the following day, sections were washed for 30 minutes in 2X SSC at room temperature and 50% formamide/2X SSC at 55°C for 30 minutes, treated in RNAse A (50 μg/ml), and dehydrated in graded ethanols. Slides were apposed to x-ray film for 28 days. A commercial polyclonal goat IgG Ab, directed against an epitope corresponding to an amino acid sequence mapping at the carboxy terminus of CCR5 of mouse origin (conserved in rat CCR5) was used; the Ab does not cross-react with other known C-C chemokine receptor gene-encoded proteins. To prepare hippocampal protein extracts, brains were rapidly removed and microdissected on ice; tissue from three animals was pooled for each sample. In addition, for preliminary experiments, samples were prepared from each cerebral hemisphere. Tissue extracts were prepared from left and right hippocampus or left and right hemispheres of animals that had received right intrahippocampal injections of NMDA (10 nmol) or PBS 32 hours earlier; samples were also collected from unlesioned P8 animals. Adult rat spleen extracts (a rich tissue source of chemokine receptors) were also prepared for use as positive controls. Samples were homogenized in 500 μl of buffer (PBS/0.1% Nonidet P-40/0.1% SDS/0.5% deoxycholic acid) containing aprotinin (5.7 μg/ml) and sodium orthovanadate (1 mmol/L); phenylmethylsulfonyl fluoride (100 μg/ml) was added after homogenization. Samples were then centrifuged (15,000 × g for 20 minutes), and supernatants were collected and stored at −20°C. Sample protein content was measured using a BCA protein assay kit according to the manufacturer's directions. Equal amounts of protein (20 μg) were resolved by SDS/10% polyacrylamide gel electrophoresis. Protein was electrotransferred to nitrocellulose in Tris/glycine buffer containing 20% methanol. Membranes were blocked overnight at 4°C in Tris-buffered saline (TBS) containing 5% gelatin and 0.2% Tween-20 and then incubated with goat anti-mouse CCR5 Ab (1:1000 in TBS/3% gelatin for 1 hour at room temperature). Membranes were washed (TBS and 0.05% Tween-20) and incubated with horseradish-peroxidase-conjugated donkey anti-goat IgG (1:15,000 in 3% gelatin in TBS), and signal was developed using enhanced luminol according to the manufacturer's directions. To prepare samples for immunocytochemistry, euthanized animals were perfused transcardially with 10 ml of PBS, followed by 10 ml of 2% paraformaldehyde in PBS. Brains were removed intact and cryoprotected in 20% sucrose as previously described;5Ivacko JA Sun R Silverstein FS Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats.Pediatr Res. 1996; 39: 1-9Crossref PubMed Scopus (135) Google Scholar14-μm frozen coronal sections were mounted onto gelatin-coated slides. Samples were prepared from animals that had received right intrahippocampal injections of 10 nmol of NMDA 24 hours (n = 2), 32 hours (n = 5), 48 hours (n = 2), 72 hours (n = 2), or 5 days (n = 2) earlier or PBS (n = 2) 32 hours earlier; two samples from unlesioned P8 animals were also assayed. At least 12 sections/brain (all including the hippocampus) were assayed. The same anti-CCR5 Ab used for the Western blot assays was also used to detect CCR5 immunocytochemically. In addition, to facilitate identification of cells of the microglia or monocyte lineage that expressed CCR5, representative adjacent sections were probed with ED-1 Ab, a MAb that recognizes a cell surface antigen expressed by activated macrophages/monocytes.5Ivacko JA Sun R Silverstein FS Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats.Pediatr Res. 1996; 39: 1-9Crossref PubMed Scopus (135) Google Scholar In addition, to identify reactive astrocytes, GFAP immunocytochemistry was performed (using a polyclonal anti-bovine GFAP Ab that detects rat GFAP). For CCR5, ED-1, and GFAP immunocytochemistry, sections were washed (in PBS for 5 minutes) and preincubated in normal horse serum (CCR5 and ED-1) or normal goat serum (GFAP) in PBS; all wash and dilution buffers for ED-1 and GFAP immunocytochemistry contained 0.1% Triton X-100. Sections were then incubated with the selected primary Ab (goat anti-CCR5, 1:100 dilution; mouse anti-ED-1, 1:500; rabbit anti-GFAP, 1:500) for 18 hours at 4°C. Equal amounts of isotype-matched IgG were substituted for the primary Ab in control samples (CCR5-goat IgG; ED-1-mouse IgG; GFAP-rabbit IgG). Sections were washed and incubated with the appropriate biotinylated secondary Ab (CCR5-horse anti-goat, 1:500; ED-1-rat adsorbed horse anti-mouse, 1:200; GFAP-goat anti-rabbit, 1:80). Sections were washed again, endogenous peroxidase activity was blocked (0.3% hydrogen peroxide in methanol for 10 minutes at room temperature), and a Vectastain ABC Elite kit was used to amplify the signal, followed by chromogenic detection with stable DAB. Sections were counterstained in 0.5% cresyl violet, dehydrated in graded ethanols, and coverslipped with Permount. To verify the specificity of CCR5 immunoreactivity, the CCR5 primary Ab was preincubated with a CCR5 blocking peptide; then the preadsorbed Ab was used under the same conditions as the CCR5 primary Ab. Intrahippocampal injection of 10 nmol of NMDA in P7 rats elicits reproducible focal neuronal loss and atrophy that is maximal in the CA3 subfield of the hippocampus; injury extends both in an antero-posterior plane from the injection site within the hippocampus and also extends to the adjacent thalamus and posterior striatum.10McDonald JW Silverstein FS Johnston MV Neurotoxicity of N-methyl-d-aspartate is markedly enhanced in developing rat central nervous system.Brain Res. 1988; 459: 200-203Crossref PubMed Scopus (443) Google Scholar At 24 hours after injury, e
Referência(s)