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Role of the Macrophage Inflammatory Protein-1α/CC Chemokine Receptor 5 Signaling Pathway in the Neuroinflammatory Response and Cognitive Deficits Induced by β-Amyloid Peptide
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.081113
ISSN1525-2191
AutoresGiselle F. Passos, Cláudia P. Figueiredo, Rui Daniel Prediger, Pablo Pandolfo, Filipe Silveira Duarte, Rodrigo Medeiros, João Β. Calixto,
Tópico(s)Chemokine receptors and signaling
ResumoThe hallmarks of Alzheimer’s disease include the deposition of β-amyloid (Aβ), neuroinflammation, and cognitive deficits. The accumulation of activated glial cells in cognitive-related areas is critical for these alterations, although little is known about the mechanisms driving this event. Herein we used macrophage inflammatory protein-1α (MIP-1α−/−)- or CC-chemokine receptor 5 (CCR5−/−)-deficient mice to address the role played by chemokines in molecular and behavioral alterations induced by Aβ1–40. Aβ1–40 induced a time-dependent increase of MIP-1α mRNA followed by accumulation of activated glial cells in the hippocampus of wild-type mice. MIP-1α−/− and CCR5−/− mice displayed reduced astrocytosis and microgliosis in the hippocampus after Aβ1–40 administration that was associated with decreased expression of cyclooxygenase-2 and inducible nitric oxide synthase, as well as reduced activation of nuclear factor-κB, activator protein-1 and cyclic AMP response element-binding protein. Furthermore, MIP-1α−/− and CCR5−/− macrophages showed impaired chemotaxis in vitro, although cytokine production in response to Aβ1–40 was unaffected. Notably, the cognitive deficits and synaptic dysfunction induced by Aβ1–40 were also attenuated in MIP-1α−/− and CCR5−/− mice. Collectively, these results indicate that the MIP-1α/CCR5 signaling pathway is critical for the accumulation of activated glial cells in the hippocampus and, therefore, for the inflammation and cognitive failure induced by Aβ1–40. Our data suggest MIP-1α and CCR5 as potential therapeutic targets for Alzheimer’s disease treatment. The hallmarks of Alzheimer’s disease include the deposition of β-amyloid (Aβ), neuroinflammation, and cognitive deficits. The accumulation of activated glial cells in cognitive-related areas is critical for these alterations, although little is known about the mechanisms driving this event. Herein we used macrophage inflammatory protein-1α (MIP-1α−/−)- or CC-chemokine receptor 5 (CCR5−/−)-deficient mice to address the role played by chemokines in molecular and behavioral alterations induced by Aβ1–40. Aβ1–40 induced a time-dependent increase of MIP-1α mRNA followed by accumulation of activated glial cells in the hippocampus of wild-type mice. MIP-1α−/− and CCR5−/− mice displayed reduced astrocytosis and microgliosis in the hippocampus after Aβ1–40 administration that was associated with decreased expression of cyclooxygenase-2 and inducible nitric oxide synthase, as well as reduced activation of nuclear factor-κB, activator protein-1 and cyclic AMP response element-binding protein. Furthermore, MIP-1α−/− and CCR5−/− macrophages showed impaired chemotaxis in vitro, although cytokine production in response to Aβ1–40 was unaffected. Notably, the cognitive deficits and synaptic dysfunction induced by Aβ1–40 were also attenuated in MIP-1α−/− and CCR5−/− mice. Collectively, these results indicate that the MIP-1α/CCR5 signaling pathway is critical for the accumulation of activated glial cells in the hippocampus and, therefore, for the inflammation and cognitive failure induced by Aβ1–40. Our data suggest MIP-1α and CCR5 as potential therapeutic targets for Alzheimer’s disease treatment. Alzheimer’s disease (AD) is the most prevalent cause of dementia in humans, and the symptoms are commonly manifested after the seventh decade of life. Numerous pathological changes have been described in the postmortem brains of AD patients, including senile plaques, tangles, neuroinflammation, synapse loss, and neuronal death.1Walsh DM Selkoe DJ Deciphering the molecular basis of memory failure in Alzheimer's disease.Neuron. 2004; 44: 181-193Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar Activated glial cells surrounding senile plaques seem to be responsible for the ongoing neuroinflammatory process in the disease through the release of cytokines and other toxic products, including reactive oxygen species, nitric oxide, and excitatory amino acids.2González-Scarano F Baltuch G Microglia as mediators of inflammatory and degenerative diseases.Annu Rev Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (895) Google Scholar However, little is known about the identity of the agent(s) responsible for glial cell accumulation and activation in the AD brain. Chemokines belong to a family of chemoattractant cytokines that were initially identified according to their ability to regulate leukocyte trafficking during inflammatory responses.3Luster AD Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3267) Google Scholar, 4Charo IF Ransohoff RM The many roles of chemokines and chemokine receptors in inflammation.N Engl J Med. 2006; 354: 610-621Crossref PubMed Scopus (2039) Google Scholar More recently, in addition to their chemotactic activity, chemokines have been implicated in the modulation of cell adhesion, phagocytosis, cytokine secretion, proliferation, apoptosis, angiogenesis, and viral pathogenesis.5Cartier L Hartley O Dubois-Dauphin M Krause KH Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases.Brain Res Brain Res Rev. 2005; 48: 16-42Crossref PubMed Scopus (408) Google Scholar In the central nervous system (CNS), these proteins regulate leukocyte migration across the brain endothelium as well as the activation and movement of cells within the brain parenchyma.6Baggiolini M Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2404) Google Scholar There is growing evidence supporting the view that resident CNS cells have the capacity to express chemokines and their receptors during a variety of neuroinflammatory and degenerative conditions.7Ransohoff RM Tani M Glabinski AR Chernosky A Krivacic K Peterson JW Chien HF Trapp BD Chemokines and chemokine receptors in model neurological pathologies: molecular and immunocytochemical approaches.Methods Enzymol. 1997; 287: 319-348Crossref PubMed Scopus (0) Google Scholar, 8Jiang 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 (141) Google Scholar, 9Simpson JE Newcombe J Cuzner ML Woodroofe MN Expression of monocyte chemoattractant protein-1 and other β-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions.J Neuroimmunol. 1998; 84: 238-249Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 10Westmoreland SV Rottman JB Williams KC Lackner AA Sasseville VG Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis.Am J Pathol. 1998; 152: 659-665PubMed Google Scholar, 11Xia MQ Qin SX Wu LJ Mackay CR Hyman BT Immunohistochemical study of the β-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains.Am J Pathol. 1998; 153: 31-37Abstract Full Text Full Text PDF PubMed Google Scholar, 12McManus C Berman JW Brett FM Staunton H Farrell M Brosnan CF MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study.J Neuroimmunol. 1998; 86: 20-29Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 13Klein RS Williams KC Alvarez-Hernandez X Westmoreland S Force T Lackner AA Luster AD Chemokine receptor expression and signaling in macaque and human fetal neurons and astrocytes: implications for the neuropathogenesis of AIDS.J Immunol. 1999; 163: 1636-1646Crossref PubMed Google Scholar, 14Liu JX Cao X Tang YC Liu Y Tang FR CCR7, CCR8, CCR9 and CCR10 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus.J Neurochem. 2007; 100: 1072-1088Crossref PubMed Scopus (40) Google Scholar Notably, recent evidence has indicated that chemokines and their receptors are up-regulated in the AD brain and that they may play a critical role in controlling the recruitment and accumulation of glial cells at β-amyloid (Aβ) sites in senile plaques.15Akiyama H Barger S Barnum S Bradt B Bauer J Cole GM Cooper NR Eikelenboom P Emmerling M Fiebich BL Finch CE Frautschy S Griffin WS Hampel H Hull M Landreth G Lue L Mrak R Mackenzie IR McGeer PL O'Banion MK Pachter J Pasinetti G Plata-Salaman C Rogers J Rydel R Shen Y Streit W Strohmeyer R Tooyoma I Van Muiswinkel FL Veerhuis R Walker D Webster S Wegrzyniak B Wenk G Wyss-Coray T Inflammation and Alzheimer's disease.Neurobiol Aging. 2000; 21: 383-421Crossref PubMed Scopus (3718) Google Scholar Macrophage inflammatory protein (MIP)-1α (CCL3) is a member of the β-chemokine subfamily, which also includes MIP-1β (CCL4) and regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5). These molecules exert their effects though activation of CC-chemokine receptor 5 (CCR5).4Charo IF Ransohoff RM The many roles of chemokines and chemokine receptors in inflammation.N Engl J Med. 2006; 354: 610-621Crossref PubMed Scopus (2039) Google Scholar CCR5 is expressed at low levels in the normal brain, but it can be induced to play important roles in various injuries or infections, including AD.5Cartier L Hartley O Dubois-Dauphin M Krause KH Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases.Brain Res Brain Res Rev. 2005; 48: 16-42Crossref PubMed Scopus (408) Google Scholar In this context, immunohistochemical analyses have revealed the expression of CCR5, together with its ligands, on the microglia of both normal and AD brains, with increased expression on some reactive microglia in AD.11Xia MQ Qin SX Wu LJ Mackay CR Hyman BT Immunohistochemical study of the β-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains.Am J Pathol. 1998; 153: 31-37Abstract Full Text Full Text PDF PubMed Google Scholar Nevertheless, the precise role of CCR5 and its ligand MIP-1α in AD is poorly understood so far. In the current study we have investigated the molecular and behavioral alterations induced by a single intracerebroventricular (i.c.v.) injection of Aβ1–40 peptide in mice lacking MIP-1α or CCR5. Although unable to induce pathological AD hallmarks, the acute injection of Aβ peptides into the rodent brain is a useful experimental model for the characterization of Aβ toxicity, as it induces an inflammatory response associated with deficits of learning and memory.16Van Dam D De Deyn PP Drug discovery in dementia: the role of rodent models.Nat Rev Drug Discov. 2006; 5: 956-970Crossref PubMed Scopus (187) Google Scholar Using this model, we demonstrated that activation of the MIP-1α/CCR5 signaling pathway is one of the earliest events after Aβ1–40 injection in mice, representing an important signal for the accumulation of activated glial cells and, consequently, for inflammatory response, synaptic dysfunction, and cognitive failure. These findings raise the possibility that MIP-1α and CCR5 represent promising targets for AD drug development. Experiments were conducted using male C57BL/6 CCR5 knockout (CCR5−/−) and MIP-1α knockout (MIP-1α−/−) mice (20 to 30 g). They were kept in controlled room temperature (22 ± 2°C) and humidity (55 to 60%) under a 12 hour light/dark cycle (lights on at 6:00 AM). CCR5−/−17Sato N Kuziel WA Melby PC Reddick RL Kostecki V Zhao W Maeda N Ahuja SK Ahuja SS Defects in the generation of IFN-γ are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1α-, or CCR2-deficient mice.J Immunol. 1999; 163: 5519-5525Crossref PubMed Google Scholar and MIP-1α−/−18Cook DN Beck MA Coffman TM Kirby SL Sheridan JF Pragnell IB Smithies O Requirement of MIP-1α for an inflammatory response to viral infection.Science. 1995; 269: 1583-1585Crossref PubMed Scopus (564) Google Scholar mice are on the C57BL/6 background, constructed as described previously. All procedures used in the present study followed the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23) and were approved by the Animal Ethics Committee of the Universidade Federal de Santa Catarina. Human Aβ1–40 (Tocris, Ellisville, MO) and the inverse peptide Aβ40–1 (Bachem, Torrance, CA) were prepared as stock solutions at a concentration of 1 mg/ml in sterile 0.1 mol/L PBS (pH 7.4), and aliquots were stored at −20°C. Aβ solutions were aggregated by incubation at 37°C for 4 days before use as described previously.19El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to β-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar The aggregation and/or oligomerization state of Aβ solutions was confirmed through Western blot analysis (data not shown). The aggregated form of Aβ fragments (400 pmol/mice) or PBS (vehicle) was administered i.c.v. as described previously.20Medeiros R Prediger RD Passos GF Pandolfo P Duarte FS Franco JL Dafre AL Di Giunta G Figueiredo CP Takahashi RN Campos MM Calixto JB Connecting TNF-α signaling pathways to iNOS expression in a mouse model of Alzheimer's disease: relevance for the behavioral and synaptic deficits induced by amyloid β protein.J Neurosci. 2007; 27: 5394-5404Crossref PubMed Scopus (261) Google Scholar, 21Prediger RD Franco JL Pandolfo P Medeiros R Duarte FS Di Giunta G Figueiredo CP Farina M Calixto JB Takahashi RN Dafre AL Differential susceptibility following β-amyloid peptide-(1–40) administration in C57BL/6 and Swiss albino mice: evidence for a dissociation between cognitive deficits and the glutathione system response.Behav Brain Res. 2007; 177: 205-213Crossref PubMed Scopus (0) Google Scholar, 22Prediger RD Medeiros R Pandolfo P Duarte FS Passos GF Pesquero JB Campos MM Calixto JB Takahashi RN Genetic deletion or antagonism of kinin B1 and B2 receptors improves cognitive deficits in a mouse model of Alzheimer's disease.Neuroscience. 2008; 151: 631-643Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar In brief, the animals were anesthetized with isoflurane (2.5%, Abbott Laboratórios do Brasil Ltda., Rio de Janeiro, RJ, Brazil) using a vaporizer system (SurgiVet Inc., Waukesha, WI) and then gently restrained by hand for i.c.v. injections. The injection site was sterilized using gauze embedded in 70% ethanol. Under light anesthesia (ie, just that necessary for loss of the postural reflex), the needle was inserted unilaterally 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eyes (used as external reference). A volume of 3 μl of Aβ1–40, Aβ40–1, or PBS solution was injected into the lateral ventricle, at the following coordinates from bregma: anteroposterior −0.22 mm, mediolateral 1 mm, and dorsoventral = −3 mm. The accurate placement of the injection site (needle track) was confirmed at the moment of dissection of the animals for molecular biology experiments. The administration site was also confirmed in parallel experiments performed by the same technician in which 2 μl of Evans blue dye 0.5% was injected, and the brains were examined microscopically to verify the staining in the walls of the lateral ventricle (more details in Ref. 22Prediger RD Medeiros R Pandolfo P Duarte FS Passos GF Pesquero JB Campos MM Calixto JB Takahashi RN Genetic deletion or antagonism of kinin B1 and B2 receptors improves cognitive deficits in a mouse model of Alzheimer's disease.Neuroscience. 2008; 151: 631-643Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Results from mice presenting misplacement of the cannula or any sign of cerebral hemorrhage were excluded from the statistical analysis (overall, less than 5% of the total animals used). To confirm the involvement of CCR5 in the glial cell accumulation induced by Aβ1–40 i.c.v. injection, some mice were treated with the CCR5 antagonist d-Ala-peptide T-amide (0.01 mg/kg/day s.c., Tocris) administered 1 hour before Aβ1–40 i.c.v. injection and throughout consecutive days until the day of the experiment. For RT-PCR and Western blot experiments, the animals were sacrificed by decapitation, and the skin was removed from the skull with two forceps. Then, one blade of a pair of fine scissors was introduced into the foramen magnum, and the skull was opened by cutting along its caudal to rostral axis. Two cuts were made perpendicular to the first one, with the scissors pointing toward the left and right ears, respectively. The opening of the skull was carefully enlarged with fine forceps. The brain was removed from the skull by means of a spatula, and the brainstem was separated from the cortex. In the next step, the hippocampuses were rapidly dissected on dry ice and stored at −70°C. For immunohistochemical studies, mice were anesthetized with chloral hydrate (400 mg/kg i.p.) and perfused transcardially with heparin (1000 U/ml) in physiological saline followed by 4% paraformaldehyde in physiological saline. The brains were rapidly removed and postfixed overnight at 4°C in 4% paraformaldehyde. Total RNA was extracted from the hippocampus using TRIzol reagent (Invitrogen, São Paulo, SP, Brazil) according to the manufacturer’s instructions. Two micrograms of total RNA was reverse transcribed using oligo(dT) as a primer (0.05 μg), 50 U of reverse transcriptase (Promega, Madison, WI), dNTP (144 μmol/L, Promega), reaction buffer (10 mmol/L dithiothreitol, 3 mmol/L MgCl2, 75 mmol/L KCl, and 50 mmol/L Tris-HCl, pH 8.3), and 2 U of RNasin Plus (Promega) in a final volume of 12.5 μl. The cDNA was obtained after incubation of the samples at 70°C for 5 minutes, 4°C for 5 minutes, 37°C for 60 minutes, 70°C for 5 minutes, and 4°C for 5 minutes. Specific primers were used for CCR5 (sense, 5′-GCCAGAGGAGGTGAGACATC-3′; antisense, 5′-AAGAGCAGGTCAGAGATGGC-3′), MIP-1α (sense, 5′-ATGAAGGTCTCCACCACTG-3′; antisense, 5′-GCATTCAGTTCCAGGTCA-3′), and β-actin (sense, 5′-TCCTTCGTTGCCGGTCCACA-3′; antisense, 5′-CGTCTCCGGAGTCCATCACA-3′). β-Actin cDNA was used for standardization of the amount of RNA. Two microliters of RT aliquots was mixed in a buffer containing 10 mmol/L Tris-HCl, pH 9, 1 mmol/L MgCl2, 200 μmol/L dNTP, 300 nmol/L of each primer, and 5 U of Taq polymerase (Ludwig Biotec, Porto Alegre, RS, Brazil) in a final volume of 30 μl. The PCR cycling protocols were as follows: initial denaturation at 95°C for 4 minutes; cycling at 95°C for 30 seconds, 54°C for 30 seconds (CCR5 and MIP-1α), or 62°C for 30 seconds (β-actin), and 72°C for 1 minute; and a final extension period at 72°C for 5 minutes. Optimal amplification was achieved at 30 cycles. Aliquots of 5 μl of each sample were analyzed by polyacrylamide gel electrophoresis and stained with silver nitrate. Band density measurements for CCR5, MIP-1α, and β-actin mRNAs were made using ImageJ 1.36b imaging software (NIH, Bethesda, MD). Tissues were homogenized in ice-cold 10 mmol/L HEPES (pH 7.4), containing 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 10 μg/ml aprotinin, 1 mmol/L sodium orthovanadate, 10 mmol/L β-glycerophosphate, 50 mmol/L sodium fluoride, and 0.5 mmol/L dithiothreitol (all from Sigma-Aldrich, St. Louis, MO). The homogenates were chilled on ice for 15 minutes, vigorously shaken for 15 minutes in the presence of 0.1% Triton X-100 and then centrifuged at 10,000 × g for 30 minutes. The supernatant containing the cytosolic fraction was stored at −70°C until use. Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then were transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore, Danvers, MA). The membranes were saturated by incubation with 10% nonfat dry milk solution and then incubated overnight with inducible nitric oxide synthase (iNOS) (1:1000), cyclooxygenase (COX)-2 (1:1000), or β-actin (1:5000) antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA). After washing, the membranes were incubated with adjusted secondary antibody coupled to horseradish peroxidase. The immunocomplexes were visualized using the an ECL chemiluminescence detection system (GE Healthcare, São Paulo, SP, Brazil). Band density measurements were made using ImageJ 1.36b. Immunohistochemical analysis was performed on paraffin-embedded brain tissue sections using CD68 (1:100, Abcam, Cambridge, MA), synaptophysin (1:400, Novocastra, Newcastle, UK), glial fibrillary acid protein (GFAP) (1:600), phospho (p)-p65 nuclear factor-κB (NF-κB; 1:100), p-cAMP-response element-binding (CREB; 1:200), p-c-Jun/activator protein-1 (AP-1) (1:300), or COX-2 (1:200) antibodies (Cell Signaling Technology, Danvers, MA). After quenching of endogenous peroxidase with 1.5% hydrogen peroxide in methanol (v/v) for 20 minutes, high-temperature antigen retrieval was performed by immersion of the slides in a water bath at 95 to 98°C in 10 mmol/L trisodium citrate buffer, pH 6.0, for 45 minutes. The slides were then processed using the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. After the appropriate biotinylated secondary antibody, sections were developed with 3,3′-diaminobenzidine (Dako Cytomation, Glostrup, Denmark) in chromogen solution for the exact amount of time and counterstained with Harris’s hematoxylin. Control and experimental tissues were placed on the same slide and processed under the same conditions. The immunostaining was assessed at four levels of the dorsal hippocampus. Specifically, four alternate 3-μm sections of the hippocampus with an individual distance of ∼150 μm were obtained between 1.6 and 2.4 mm posterior to the bregma. Images of stained hippocampal CA1, CA2, and CA3 and dentate gyrus subregions were acquired using a Sight DS-5M-L1 digital camera (Nikon, Melville, NY) connected to an Eclipse 50i light microscope (Nikon). A threshold optical density that best discriminated staining from the background was obtained using the ImageJ 1.36b. We captured two images of each hippocampal subregion per section (8 images per section and 32 images per mouse). For p-CREB or COX-2 burden analyses, data are reported as the percentage of labeled area captured (positive pixels) divided by the full area captured (total pixels), as described previously.23Town T Laouar Y Pittenger C Mori T Szekely CA Tan J Duman RS Flavell RA Blocking TGF-β-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology.Nat Med. 2008; 14: 681-687Crossref PubMed Scopus (365) Google Scholar For synaptophysin analysis, total pixel intensity was determined, and data were expressed as optical density. The data represent the average value obtained by the analysis of images of the hippocampal CA1, CA2, and CA3 and dentate gyrus subregions. The numbers of CD68, GFAP, p-p65 NF-κB, or p-c-Jun AP-1 stained-positive cells were examined microscopically at ×40 magnification. The numbers of stained-positive cells within the CA1, CA2, CA3, and dentate gyrus subregions of each of the four 3-μm sections were counted. The mean number of stained-positive cells per section was calculated for each animal group. All histological assessments were made by an examiner blinded to sample identities. Some inherent weaknesses of the two-dimensional counting and densitometry methods applied in this study have been reviewed elsewhere.24Benes FM Lange N Two-dimensional versus three-dimensional cell counting: a practical perspective.Trends Neurosci. 2001; 24: 11-17Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar For the immunofluorescence experiments, brains were sectioned at 40 μm using a Vibratome (Pelco, Redding, CA). Sections were first blocked with 3% normal serum with 2% bovine serum albumin and 0.1% Triton X-100 in Tris-buffered saline and then were incubated with ionized calcium binding adaptor molecule-1 (Iba-1) primary antibody (1:200) (Wako Chemicals, Richmond, VA) in blocking solution overnight at 4°C. Sections were then rinsed and incubated for 1 hour with secondary Alexa Fluor-conjugated antibody (Invitrogen) at room temperature. Finally, sections were mounted onto gelatin-coated slides in Fluoromount-G (Southern Biotech, Birmingham, AL) and examined under a confocal laser microscope (Olympus, Tokyo, Japan) using Laser Sharp 2000 software (Bio-Rad). The number of Iba-1-positive cells was examined microscopically at ×40 magnification at four levels of the dorsal hippocampus with an individual distance of ∼150 μm, obtained between 1.6 and 2.4 mm posterior to the bregma. The numbers of stained-positive cells within the CA1, CA2, CA3, and dentate gyrus subregions of each of the four sections were counted. The mean number of stained-positive cells per section was calculated for each animal group. Resident peritoneal macrophages were harvested from wild-type (C57BL/6), CCR5−/−, or MIP-1α−/− mice. Cells were incubated on 24-well cell culture plates (106 cells/well) in RPMI 1640 containing 5% fetal calf serum, 2 mmol/L l-glutamine, 150 U/ml penicillin, and 150 μg/ml streptomycin (Invitrogen) at 37°C and were stimulated with Aβ1–40 or Aβ40–1 (30 μmol/L). After 24 hours the supernatant was harvested and stored at −80°C. The chemotaxis assays were performed with a chemotaxis instrument (Neuro Probe, Inc., Gaithersburg, MD). In brief, 27 μl of supernatant from wild-type, CCR5−/−, or MIP-1α−/− macrophages, stimulated with Aβ1–40 or Aβ40–1, was placed in the lower compartment of the chemotaxis chamber, and 50 μl of 3 × 106 cells/ml (obtained from wild-type, CCR5−/−, or MIP-1α−/− mice) was placed in the upper compartment. The two compartments were separated by a porous polycarbonate filter (5-μm pore size). Macrophages were allowed to migrate for 2 hours at 37°C, and the number of cells that had migrated to the lower surface of the filter was determined using a light microscope. The cells were counted in the May-Grünwald-Giemsa-stained filters under oil immersion magnification in 20 consecutive noncoincident fields. The chemotaxis index represents the number of cells that migrated in response to supernatant from stimulated macrophages divided by the number of cells that migrated in response to supernatant from unstimulated macrophages. Each data point is the result of triplicate measurements, from a single representative experiment. Each experiment was repeated three times with similar results. Resident peritoneal macrophages were obtained from wild-type, CCR5−/−, or MIP-1α−/− mice and were stimulated with Aβ1–40 (30 μmol/L), Aβ40–1 (30 μmol/L), Escherichia coli lipopolysaccharide (LPS) (serotype 0111:B4, 100 ng/ml, Sigma-Aldrich), or complement component C5a (10 nmol/L, Sigma-Aldrich) for 24 hours. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in supernatants were measured using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). The cell viability of the stimulated macrophages was determined by mitochondria-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich) to formazan as described previously.25Mosmann T Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Methods. 1983; 65: 55-63Crossref PubMed Scopus (46770) Google Scholar The Morris water maze test was performed as described previously.26Morris RG Garrud P Rawlins JN O'Keefe J Place navigation impaired in rats with hippocampal lesions.Nature. 1982; 297: 681-683Crossref PubMed Scopus (5106) Google Scholar The experimental apparatus consisted of a circular tank (diameter 97 cm; height 60 cm) containing water at 22 ± 2°C. The target platform (10 × 10 cm) was submerged 1 cm below the surface and placed at the midpoint of one quadrant. The platform was located in a fixed position, equidistant from the center and the wall of the tank. The tank was located in a test room containing various prominent visual cues. Mice were submitted to a spatial reference memory version of the water maze as described previously.20Medeiros R Prediger RD Passos GF Pandolfo P Duarte FS Franco JL Dafre AL Di Giunta G Figueiredo CP Takahashi RN Campos MM Calixto JB Connecting TNF-α signaling pathways to iNOS expression in a mouse model of Alzheimer's disease: relevance for the behavioral and synaptic deficits induced by amyloid β protein.J Neurosci. 2007; 27: 5394-5404Crossref PubMed Scopus (261) Google Scholar, 21Prediger RD Franco JL Pandolfo P Medeiros R Duarte FS Di Giunta G Figueiredo CP Farina M Calixto JB Takahashi RN Dafre AL Differential susceptibility following β-amyloid peptide-(1–40) administration in C57BL/6 and Swiss albino mice: evidence for a dissociation between cognitive deficits and the glutathione system response.Behav Brain Res. 2007; 177: 205-213Crossref PubMed Scopus (0) Google Scholar, 22Prediger RD Medeiros R Pandolfo P Duarte FS Passos GF Pesquero JB Campos MM Calixto JB Takahashi RN Genetic deletion or antagonism of kinin B1 and B2 receptors improves cognitive deficits in a mouse model of Alzheimer's disease.Neuroscience. 2008; 151: 631-643Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar The acquisition training session was performed 7 days after Aβ1–40 injection and consisted of 10 consecutive trials, during which the animals were left in the tank facing the wall and allowed to swim freely to the escape platform. If an animal did not find the platform within a period of 60 seconds, it was gently guided to it. The animal was allowed to remain on the platform for 10 seconds after escaping to it and was then removed from the tank for 5 minutes before being placed
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