Chronic Neuron-Specific Tumor Necrosis Factor-Alpha Expression Enhances the Local Inflammatory Environment Ultimately Leading to Neuronal Death in 3xTg-AD Mice
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080528
ISSN1525-2191
AutoresMichelle C. Janelsins, Michael A. Mastrangelo, Keigan M. Park, Kelly L. Sudol, Wade C. Narrow, Salvatore Oddo, Frank M. LaFerla, Linda M. Callahan, Howard J. Federoff, William J. Bowers,
Tópico(s)Immune Response and Inflammation
ResumoInflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimer's disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-α, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-α in 3xTg-AD mice. To further define the role of neuronally derived TNF-α during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-α was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-β and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-α and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-α expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-α-specific agents to subvert AD. Inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimer's disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-α, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-α in 3xTg-AD mice. To further define the role of neuronally derived TNF-α during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-α was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-β and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-α and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-α expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-α-specific agents to subvert AD. Inflammation has long been hypothesized to play a critical role in Alzheimer's disease (AD).1Billings LM Oddo S Green KN McGaugh JL LaFerla FM Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice.Neuron. 2005; 45: 675-688Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 2Griffin WS Inflammation and neurodegenerative diseases.Am J Clin Nutr. 2006; 83: 470S-474SPubMed Google Scholar, 3Streit WJ Microglia and Alzheimer's disease pathogenesis.J Neurosci Res. 2004; 77: 1-8Crossref PubMed Scopus (288) Google Scholar Focal and diffuse gliosis is highly evident in areas of pathology, especially at sites of ghost tangles, amyloid-bearing plaques, and angiopathic capillaries in late-stage AD brain.4Duffy PE Rapport M Graf L Glial fibrillary acidic protein and Alzheimer-type senile dementia.Neurology. 1980; 30: 778-782Crossref PubMed Google Scholar, 5Haga S Akai K Ishii T Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain. 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An immunocytochemical and electron-microscopic study.Acta Neuropathol (Berl). 1982; 57: 75-79Crossref PubMed Scopus (108) Google Scholar Correlating with pathology, steady-state levels of inflammatory molecules, including tumor necrosis factor-α (TNF-α), interleukin-1beta, and complement components, are significantly enhanced in postmortem brain tissue and cerebrospinal fluid from AD-afflicted individuals (reviewed in9McGeer PL McGeer EG Local neuroinflammation and the progression of Alzheimer's disease.J Neurovirol. 2002; 8: 529-538Crossref PubMed Scopus (227) Google Scholar). While extant data better support a secondary role for inflammation in AD pathogenesis rather than an etiological one, the precise function of inflammatory processes, especially those initiated at presymptomatic stages, has yet to be elucidated. The cells of the central nervous system known to produce pro-inflammatory mediators, such as TNF-α, in response to AD-related insults include astrocytes, microglia, and neurons (reviewed in10Strohmeyer R Rogers J Molecular and cellular mediators of Alzheimer's disease inflammation.J Alzheimers Dis. 2001; 3: 131-157PubMed Google Scholar). Although microglia and astrocytes are classically believed to serve as the predominant sources of TNF-α in the central nervous system, neurons can highly express this cytokine in the setting of disease, including spinal cord injury,11Ohtori S Takahashi K Moriya H Myers RR TNF-alpha and TNF-alpha receptor type 1 up-regulation in glia and neurons after peripheral nerve injury: studies in murine DRG and spinal cord.Spine. 2004; 29: 1082-1088Crossref PubMed Scopus (297) Google Scholar stroke,12Liu T Clark RK McDonnell PC Young PR White RF Barone FC Feuerstein GZ Tumor necrosis factor-alpha expression in ischemic neurons.Stroke. 1994; 25: 1481-1488Crossref PubMed Scopus (713) Google Scholar and sciatic nerve injury.13Schafers M Geis C Svensson CI Luo ZD Sommer C Selective increase of tumour necrosis factor-alpha in injured and spared myelinated primary afferents after chronic constrictive injury of rat sciatic nerve.Eur J Neurosci. 2003; 17: 791-804Crossref PubMed Scopus (195) Google Scholar Several AD-related studies have investigated the effects of TNF-α, particularly in relation to microglia-mediated release; however, none have explored the role of neuronally derived TNF-α during early AD pathogenesis. Using the triple transgenic-AD (3xTg-AD) mouse model, which exhibits progressive temporal and regional amyloid and tau-related pathologies, we previously demonstrated that TNF-α expression and numbers of microglia are markedly enhanced at prepathological time points in the brain.14Janelsins MC Mastrangelo MA Oddo S LaFerla FM Federoff HJ Bowers WJ Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice.J Neuroinflammation. 2005; 2: 23Crossref PubMed Scopus (204) Google Scholar These inflammatory changes are coincident with the appearance of cognitive deficits and synaptic dysfunction in these mice,1Billings LM Oddo S Green KN McGaugh JL LaFerla FM Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice.Neuron. 2005; 45: 675-688Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 15Oddo S Caccamo A Shepherd JD Murphy MP Golde TE Kayed R Metherate R Mattson MP Akbari Y LaFerla FM Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3302) Google Scholar suggesting that TNF-α participates in early disease-related pathophysiology. Herein, we demonstrate that neurons in the brains of 3xTg-AD mice express TNF-α and investigate the effects that neuronally derived TNF-α impart on AD-related pathological outcome. TNF-α was constitutively expressed in the 3xTg-AD and non-transgenic (Non-Tg) mouse hippocampus beginning at 2 months of age via a recombinant adeno-associated virus serotype 2 vector (rAAV). Brain-specific effects of TNF-α overexpression on pro-inflammatory gene expression, amyloid and tauopathy progression, and neuronal viability were assessed. Recombinant AAV-mediated TNF-α expression led to defined early activation of proximal microglia and the number of neurons harboring intracellular amyloid-β (Aβ), but imparted no apparent effect on glial fibrillary acidic protein (GFAP)-positive astrocytes in 3xTg-AD mice. Following a protracted period of TNF-α overexpression, significant neuronal death as well as pronounced activation of microglia and leukocyte infiltration, were clearly evident specifically in the brains of 3xTg-AD mice, suggesting that TNF-α-related signaling cascades and the AD-related transgene products of 3xTg-AD mice cooperate in vivo to lead ultimately to neuronal death. Overall, these data point to a potentially significant role of TNF-α-directed processes in the progression of early human AD. Triple transgenic-AD (3xTg-AD) and non-transgenic (Non-Tg) mice were created as previously described.15Oddo S Caccamo A Shepherd JD Murphy MP Golde TE Kayed R Metherate R Mattson MP Akbari Y LaFerla FM Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3302) Google Scholar Six-month-old mice were used for in situ hybridization studies, while 2-month-old mice were used at the initiation of the virus vector transduction experiments. All animal housing and procedures were performed in compliance with guidelines established by the University Committee of Animal Resources at the University of Rochester. Six-month old 3xTg-AD and Non-Tg mice were sacrificed and perfused using 10% buffered neutral formalin, followed by 24-hour incubations in 10% formalin, 1× PBS, 20% sucrose, and 30% sucrose before mounting sections onto slides in RNase-free conditions. Both immunohistochemistry and in situ hybridization was performed under rigorous RNase-free conditions throughout the procedure. For immunohistochemistry, slides were incubated in 5× PBS for 15 minutes, followed by two incubations of 1× PBS for 30 minutes each. Slides were then incubated in 0.15 mol/L phosphate buffer (PB) containing 1% H2O2 for 25 minutes. Following peroxidase quenching, slides were incubated in 0.15 mol/L PB containing 0.1% Triton-X 100 for 5 minutes and then moved to blocking solution containing 0.1% Triton-X 100, 0.1% normal goat serum, 0.5% bovine serum albumin, and RNase inhibitor (RNAsin, Promega, Madison, WI) for 3 hours with gentle rotation. Primary NeuN (1:100; Chemicon, Temecula, CA) or F4/80 antibody (1:100; Serotec, Raleigh, NC) was then added in blocking solution and incubated for 36 hours at 4°C. Slides were then washed thrice, 10 minutes each, in blocking solution before 2-hour incubation with a biotinylated anti-mouse secondary antibody (1:500, Vector Labs, Burlingame, CA) in blocking solution at room temperature (∼22°C). Two 5-minute washes in 0.15 mol/L PB were used to remove excess secondary antibody. Slides were incubated in an avidin-biotinylated horseradish peroxidase complex (Vector Labs) in 0.15 mol/L PB for 1.5 hours, followed by a wash in 0.15 mol/L PB for 3 × 5 minutes and H2O for 2 × 5 minutes before diaminobenzidine (DAB) development (Vector Labs) for 15 minutes. The slides were subsequently rinsed in distilled water (dH2O) and held under RNase-free conditions in 1× PBS before proceeding to in situ hybridization. For in situ hybridization, slides were washed in 4% paraformaldehyde for 20 minutes, followed by 3× PBS, 1× PBS, 1× PBS, and dH2O for 5 minutes each. Slides were then incubated in a 37°C bath of 1 μg/ml proteinase K (Qiagen, Valencia, CA) in Tris/EDTA, pH 8.0 for 30 minutes. Slides were washed twice for 1 minute each in 1× PBS before a 10-minute fixation in 4% paraformaldehyde and then a 1-minute 0.2% glycine treatment. Slides were washed briefly before 0.25% acetic anhydride treatment in 0.1 mol/L TEA twice for 10 minutes each. Slides were subsequently washed in 1× PBS for 5 minutes, and dehydrated through ethanol, before a 20-minute incubation in chloroform. Following delipidation, slides were incubated in 100% and 95% ethanol before air-drying for 45 minutes. Anti-sense and sense TNF-α riboprobes were synthesized from a TOPO plasmid with T7 directing anti-sense and SP6 RNA polymerase directing sense riboprobe production. The mouse TNF-α probe consisted of bases 45 through 557 of the mRNA coding sequence. Riboprobe synthesis was conducted using an in vitro transcription system (Promega) and 35S-UTP (Perkin Elmer, Waltham, MA) to generate probes with a specific activity of approximately 1 × 108 cpm/μg. Excess nucleotides were removed using the RNAid clean-up kit (QbioGene, La Jolla, CA). Anti-sense and sense hybridization probes were made by adding probe to a hybridization solution containing 50% formamide, 0.3 mol/L salts solution, 0.1 mol/L dithiothreitol, and 10% dextran sulfate. Incubation at 80°C for 10 minutes was performed before incubation on ice and reheating to 56°C. Labeled probe was maintained at 56°C until addition to slides, coverslipping, DPX (Electron Microscopy Sciences, Hatfield, PA) mounting media of the coverslip edges, and placement in a hybridization chamber at 56°C for 12 hours. Coverslips were removed in 4× standard saline citrate with 0.1 mol/L dithiothreitol and then all slides were washed in 4 changes of 4× standard saline citrate with 0.1 mol/L dithiothreitol for 30 minutes each. Ethanol dehydration with 0.3 mol/L ammonium acetate was performed on slides before movement to a high stringency formamide/Tris-EDTA wash at 78°C. A 10-minute wash in 2× SSC was performed and slides were treated in a 20 μg/ml RNase A (Sigma-Aldrich, St. Louis, MO) bath. Slides were washed twice for 15 minutes each in RNase bath buffer, and once for 30 minutes in 2× standard saline citrate containing 0.01 mol/L β-mercaptoethanol. Lastly, slides were dehydrated with ethanol and dried for 1 hour before placing them against emulsion-coated film overnight. In a darkroom, slides were subsequently dipped in NTB emulsion (Kodak, Rochester, NY) at 43°C, dried for 2 hours, and placed at 4°C for 8 weeks for development. The pFBGR plasmid harbors a cytomegalovirus promoter-driven enhanced green fluorescent protein (eGFP) gene flanked by inverted terminal repeats (pAAV-eGFP, kindly provided by Dr. Robert Kotin). Human tumor necrosis factor-α (hTNF-α) cDNA from pE4 (ATCC, Manassas, VA) was cloned into the pBSFBRmcs shuttle vector and subsequently into a modified pFBGR plasmid backbone devoid of the eGFP gene. This resultant plasmid was designated pAAV-TNFα. The pAAV-eGFP and pAAV-TNFα plasmids were transiently transfected into baby hamster kidney cells and transgene expression confirmed by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) before viral packaging. The pAAV-eGFP and pAAV-TNFα plasmids were separately transposed into DH10-BAC E. coli (Invitrogen, Carlsbad, CA) and DNA purified before transfection into SF9 cells to produce baculovirus and finally AAV vector particles according to previously published methodology.16Urabe M Ding C Kotin RM Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther. 2002; 13: 1935-1943Crossref PubMed Scopus (375) Google Scholar 293A cells were transduced with either rAAV-TNFα or rAAV-eGFP to confirm TNF-α expression by enzyme-linked immunosorbent assay or to obtain titer information via flow cytometry. Titers were expressed as transducing units (TU) per ml. Recombinant AAV-TNFα or AAV-eGFP vectors or saline were stereotactically delivered into 2-month-old 3xTg-AD and Non-Tg mice in accordance with approved University of Rochester animal use guidelines. Mice were anesthetized with Avertin (300 mg/kg) and monitored throughout the stereotactic procedure to maintain a plane of surgical anesthesia. After positioning the mouse in a stereotactic apparatus (ASI Instruments, Warren, MI), the skull was exposed via a midline incision, and two burr holes were drilled bilaterally over the designated hippocampal coordinates (Bregma, −2.7 mm; lateral, 2.0 mm; ventral, 1.3 mm; and Bregma, −2.9 mm; lateral, 2.5 mm; ventral, 1.7 mm). A 33 gauge needle was gradually advanced to the desired depth during a 1.5-minute period. All injections were performed using a microprocessor controlled pump (UltraMicro-Pump; WPI Instruments, Sarasota, FL). A total of 2 μl (3.0 × 109 TU) per injection was delivered at a constant rate of 200 nl/min. A 3xTg-AD mouse cohort sacrificed at 6 months for histological analysis was injected with either rAAV-TNFα or rAAV-eGFP in the right hippocampus and saline in the left (n = 4 for rAAV-TNFα injected and n = 3 for rAAV-eGFP). To confirm results of intracellular Aβ42 immunohistochemistry (described below), a second cohort of 3xTg-AD mice was identically injected (n = 4). Non-Tg and 3xTg-AD mice (n = 6/group) sacrificed at 12 months of age for histological analysis were injected bilaterally with rAAV-TNFα and rAAV-eGFP. Mice assessed by quantitative real time reverse transcription-PCR (qRT-PCR) analysis were injected bilaterally with either rAAV-TNFα or rAAV-eGFP according to the stereotactic coordinates detailed above (n = 4/group for rAAV-TNFα injected and n = 3 for rAAV-eGFP at the 6-month time point and n = 6/group for the 12-month time point). Following each injection, the needle was extracted over a 3-minute period. Incisions were closed using vicryl sutures, topical 5% lidocaine ointment (Fougera, Melville, NY) applied, and mice were allowed to recuperate in a heated recovery chamber before returning to their housing cage and the vivarium. RNA was isolated from microdissected hippocampi of rAAV-TNFα or rAAV-eGFP mice with TRIzol solution (Invitrogen) at 6 and 12 months of age, which represent 4 and 10 months postvector infusion, respectively, as described previously.14Janelsins MC Mastrangelo MA Oddo S LaFerla FM Federoff HJ Bowers WJ Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice.J Neuroinflammation. 2005; 2: 23Crossref PubMed Scopus (204) Google Scholar One microgram of total RNA was reverse transcribed using Applied Biosystems High-Capacity cDNA Archive Kit. An aliquot of cDNA (100 ng) was used to assess the levels of 19 or 15 targets per mouse at the 6- and 12-month time points, respectively. Each sample was analyzed in a standard PE7900HT quantitative RT-PCR reaction as previously described14Janelsins MC Mastrangelo MA Oddo S LaFerla FM Federoff HJ Bowers WJ Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-1 expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice.J Neuroinflammation. 2005; 2: 23Crossref PubMed Scopus (204) Google Scholar using a TaqMan Assay-on-Demand primer-probe sets in Microfluidic cards (Applied Biosystems, Foster City, CA). An included 18S RNA primer-probe set served as the control to which all samples were normalized. The resultant data were analyzed using the ΔΔCT method, normalizing the rAAV-TNFα values to samples from rAAV-eGFP injected 3xTg-AD or Non-Tg mice, or data were normalized to Non-Tg mice when 3xTg-AD mice were compared to the Non-Tg group in rAAV-TNFα specific differences. To determine statistical significance, Student's t-test or analysis of variance with Bonferroni posthoc tests were performed, as indicated. For analysis of human APPSwe, PS1M146V, or TauP301L transgene expression and human TNF-α expression derived from the rAAV vector, 25 ng cDNA was analyzed in a 7300 quantitative PCR machine using a TaqMan Assay-on-Demand primer probe set to human APP, PS1, Tau or TNF-α (Applied Biosystems). At 6 or 12 months of age, injected 3xTg-AD and Non-Tg mice were sacrificed and brains were fixed by transcardiac perfusion with 4% paraformaldehyde in 0.1 mol/L PB. The brains were removed, postfixed overnight in 4% paraformaldehyde in 0.1 mol/L PB, transferred to a solution of 20% sucrose in PBS overnight, and subsequently placed in a solution of 30% sucrose in PBS. Immunohistochemical analysis was performed on 30-μm free-floating brain sections using the following antibodies: MAB610 at 25 μg/ml (R&D Systems, Minneapolis, MN) to detect hTNF-α; anti-GFP at 1:2000 (Invitrogen) to detect eGFP; anti-amyloid precursor protein A4, corresponding to the NPXY motif of hAPP (Clone Y188; AbCam, Cambridge, MA, 1:750); anti-hAPP/amyloid-β reactive to amino acid residues 1 to 16 of β-amyloid (6E10; Covance, Berkeley, CA; 1:1000); anti-amyloid β 1-42 clone 12F4 reactive to the C-terminus of β-amyloid and specific for the isoform ending at amino acid 42 (Covance/Signet, Berkeley, CA, 1:1000); anti-amyloid β 1-42 polyclonal antibody for intracellular amyloid-β staining (Invitrogen, formerly Biosource, Hopkinton, MA; 1:1000); AT180 (Chemicon, Temecula, CA) to detect hyperphosphorylated tau at 1:200; F4/80 at 1:500 (Serotec, Raleigh, NC) to stain for microglia/macrophages; GFAP at 1:1000 (Dako, Carpinteria, CA) for astrocytes; and NeuN at 1:500 (Chemicon) to detect mature neurons. The Aβ, APP/Aβ, AT180, F4/80, GFAP, and NeuN immunohistochemical analyses were performed using DAB development. Sections were washed three times for 5 minutes each to remove cyroprotectant, then three times for 30 minutes each in 0.15 mol/L PB. Endogenous peroxidase activity was quenched by incubation in 0.15 mol/L PB containing 3% H2O2 for 25 minutes. Sections being processed for Aβ or APP/Aβ visualization were treated at this stage with 3% methanol. Sections were subsequently washed twice for 5 minutes each with 0.15 mol/L PB. Staining with 12F4 required epitope retrieval treatment using 90% formic acid (Sigma-Aldrich) for 5 minutes at room temperature, followed by two washes for 5 minutes each with PB. Tissue was permeabilized with 0.15 mol/L PB + 0.4% Triton-X 100 (Sigma-Aldrich). Non-specific interactions were blocked by incubation of the sections for 1 hour at 22°C with 0.15 mol/L PB + 0.4% Triton-X 100 + 10% normal goat serum (Gibco/Invitrogen, Carlsbad, CA). Sections were incubated overnight at 4°C with primary antibody diluted in 0.15 mol/L PB + 0.4% Triton-X 100 + 1% normal goat serum. Samples were washed three times for 10 minutes each with 0.15 mol/L PB + 0.4% Triton-X 100 + 1% normal goat serum, before addition of biotinylated species-specific secondary antibody IgG (H+L) generated in goat (1:1000; Vector Labs), diluted in 0.15 mol/L PB + 0.4% Triton-X 100 + 1% normal goat serum. Excess secondary antibody was washed away with 0.15 mol/L PB. A conjugate was formed with bound secondary and avidin:biotinylated complex in an enzyme reaction using 2 μl of solution A and 2 μl of solution B from a Vectastain ABC kit per ml of 0.15 mol/L PB (Vector Labs). Sections were developed using a DAB peroxidase kit, according to manufacturer's instructions for nickel enhancement (Vector Labs) and mounted on slides for visualization by microscopy. Immunocytochemistry was performed for enhanced GFP or hTNF-α, or in combination with NeuN or GFAP cell markers using fluorescently labeled secondary antibodies. Tissue sections were washed with PBS to remove cyroprotectant, as above. Sections were permeabilized with PBS + 0.1% Triton-X 100 for 5 minutes and then incubated with blocking serum (PBS + 0.1% Triton-X 100 and 10% normal goat serum) for 1 hour at 22°C. Primary antibody combinations were diluted into PBS + 0.1% Triton-X 100 + 0.1% normal goat serum overnight at 4°C. Sections were washed three times 10 minutes each and then incubated in appropriate secondary antibodies for 2 hours at 22°C. Anti-mouse Alexa 647 at 1:500 (Invitrogen) was used for hTNF-α immunohistochemistry, anti-rabbit Alexa 488 at 1:500 (Invitrogen) was used for eGFP immunohistochemistry. When appropriate, preconjugated (Alexa 568 Invitrogen) NeuN or a Cy3 GFAP (Sigma-Aldrich) was then applied and sections were incubated overnight at 4°C. Sections were washed with 0.1 mol/L PBS, mounted, and coverslipped with the aqueous mounting media Mowiol. Imaging was performed using a Zeiss Scanning confocal microscope (Carl Zeiss Inc., Minneapolis, MN). For intracellular Aβ1-42 staining, a microwave/Target buffer (Dako Cytomation, Glostrup, Denmark) epitope retrieval method was used as described in17D'Andrea MR Reiser PA Polkovitch DA Gumula NA Branchide B Hertzog BM Schmidheiser D Belkowski S Gastard MC Andrade-Gordon P The use of formic acid to embellish amyloid plaque detection in Alzheimer's disease tissues misguides key observations.Neurosci Lett. 2003; 342: 114-118Crossref PubMed Scopus (45) Google Scholar; briefly the brain sections were washed with 0.15 mol/L PB for 2 hours to remove the cryoprotectant, then incubated with 3% H2O2 in 0.15 mol/L PB for 20 minutes to quench endogenous peroxidase activity before mounting sections onto slides (Superfrost Plus, VWR International, West Chester, PA). The Target buffer was heated to 98°C in a microwave (GE, Louisville, KY), and the slides submerged into the buffer and heated in the microwave, twice for 3 minutes at 450 W, and allowed to stand for 5 minutes between each microwave step. The sections were washed and permeabilized in 0.15 mol/L PB and 0.4% Triton X-100, followed by blocking in 0.15 mol/L PB + 0.4% Triton X-100 + 10% normal goat serum. After blocking, the sections were incubated in 0.15 mol/L PB + 0.4% Triton X-100 + 1% normal goat serum, with an Aβ1-42 specific primary antibody (polyclonal anti-amyloid β 1-42; Invitrogen). The sections were washed with 0.15 mol/L PB, and followed by an incubation with the appropriate secondary biotin-conjugated secondary antibody (Vector Labs; 1:1000) in 0.15 mol/L PB + 0.4% Triton X-100 + 1% normal goat serum. The sections were washed with 0.15 mol/L PB + 0.4%Triton X-100 + 1% normal goat serum, and incubated in the avidin-biotin complex (Vector Labs Vectastain ABC System as per manufacturer's protocol). Sections were washed in 0.15 mol/L PB followed by rinses in dH2O. The sections were developed with nickel-enhanced DAB (Vector Labs), dried, and coverslipped. All immunohistochemically stained sections were viewed using an Olympus AX-70 microscope, and DP71 camera, and controller software (Olympus, Center Valley, PA). Positive cells were visualized using an Olympus AX-70 microscope equipped with a motorized stage (Olympus, Melville, NY) and the SPOT camera and software (Diagnostic Instruments, Sterling Heights, MI). Following F4/80 DAB immunohistochemistry, 12-month time-point sections were incubated in Nuclear Fast Red counterstain (Vector Labs) for 30 minutes, followed by a 10-minute destaining step in dH2O, and alcohol dehydration before sealing under coverslips. Red nuclei were visualized in CA1 pyramidal and dentate gyrus (DG) granule cell layers, which predominately contain neurons, using an Olympus AX-70 microscope (Olympus, Melville, NY) connected to a SPOT camera (Diagnostic Instruments, Sterling Heights, MI). The MCID Elite 6.0 Imaging Software (Imaging Research, Inc.) was used for semiquantitative analysis. Three sections from 3.7 mm and 3.9 mm posterior from Bregma were analyzed from each region and each mouse (n = 6 for 3xTg-AD mice and n = 4 for Non-Tg mice). A defined region encompassing 175,000 μm2 was analyzed in each counting frame. All nuclei enumerated were negative for the microglial marker F4/80. CD45 immunohistochemical analysis of brain sections from rAAV vector-injected 12 month-old 3xTg-AD and Non-Tg mice was performed directly on slides. Sections were washed three times for 5 minutes each to remove cyroprotectant, then three times for 30 minutes each in 0.15 mol/L PB. Endogenous peroxidase activity was quenched by incubation in 0.15 mol/L PB containing 3% H2O2 for 25 minutes. Sections were subsequently washed twice for 5 minutes each with 0.15 mol/L PB. Tissue was permeabilized with 0.15 mol/L PB + 0.4% Triton-X 100 (Sigma-Aldrich) for 5 minutes before mounting on slides. Slides were then dried for 10 minutes on a slide warmer at 42°C and then repermeabilized for 15 minutes. Non-specific interactions were blocked by incubation of the sections for 1 hour at 22°C with PB + 0.4% PB Triton-X 100 + 10% normal goat serum (Invitrogen). Sections were incubated overnight at 4°C with 500 μl solution of anti-CD45 MCA1031G (Serotec) antibody diluted in PB + 0.4% Triton-X 100 + 1% normal goat serum. Samples were washed three times for 10 minutes each with PB + 0.4% Triton- X 100 + 1% normal goat serum before addition of biotinylated anti-rat secondary antibody IgG generated in goat (1:1000; Vector Labs), diluted i
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