Lipopolysaccharide-Induced Epithelial Monoamine Oxidase Mediates Alveolar Bone Loss in a Rat Chronic Wound Model
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090108
ISSN1525-2191
AutoresDaisuke Ekuni, James D. Firth, Tarun Nayer, Takaaki Tomofuji, Toshihiro Sanbe, Koichiro Irie, Tatsuo Yamamoto, Takashi Oka, Zhenzi Liu, Juergen R. Vielkind, Edward E. Putnins,
Tópico(s)Adenosine and Purinergic Signaling
ResumoReactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated ≥4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H2O2, as well as MAO/B and tumor necrosis factor (TNF)-α levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H2O2 production and TNF-α expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H2O2 levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H2O2 and TNF-α activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss. Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated ≥4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H2O2, as well as MAO/B and tumor necrosis factor (TNF)-α levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H2O2 production and TNF-α expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H2O2 levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H2O2 and TNF-α activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss. The production of microbicidal reactive oxygen species (ROS), a key feature of the innate immune system, has been best characterized in professional phagocytes.1Rosen H Crowley JR Heinecke JW Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis.J Biol Chem. 2002; 277: 30463-30468Crossref PubMed Scopus (99) Google Scholar Although epithelial cells are the first line of defense against bacterial challenge, the antimicrobial ROS properties of this cell type have been, to date, largely unstudied. Recently it has been shown that natural gut epithelial infection in Drosophila is associated with rapid ROS synthesis, but flies that lack normal ROS cycling capacity have increased mortality rates.2Ha E-M Oh C-T Ryu J-H Bae Y-S Kang S-W Jang I-H Brey PT Lee WJ An antioxidant system required for host protection against gut infection in Drosophila.Dev Cell. 2005; 8: 125-132Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar In the event of chronic infection, however, excessive ROS production may become toxic to the host. Gastric epithelial cells exposed to various strains of Helicobacter pylori showed a dose-dependent increase in ROS generation. Likewise ROS levels were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals.3Ding S-Z Minohara Y Fan XJ Wang J Reyes VE Patel J Dirden-Kramer B Boldogh I Ernst PB Crowe SE Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells.Infect Immun. 2007; 75: 4030-4039Crossref PubMed Scopus (161) Google ScholarH. pylori strains bearing the Cag pathogenicity island are associated with greater peptic ulceration4Blaser MJ Perez-Perez GI Kleanthous H Cover TL Peek RM Chyou PH Stemmermann GN Nomura A Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach.Cancer Res. 1995; 55: 2111-2115PubMed Google Scholar and induce higher levels of ROS and activated apoptosis markers caspase 3 and 8 than isogenic Cag pathogenicity island-deficient mutants.3Ding S-Z Minohara Y Fan XJ Wang J Reyes VE Patel J Dirden-Kramer B Boldogh I Ernst PB Crowe SE Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells.Infect Immun. 2007; 75: 4030-4039Crossref PubMed Scopus (161) Google Scholar The pathogenicity island may act by elevating mitochondrial ROS formation5Handa O Naito Y Yoshikawa T CagA protein of Helicobacter pylori: a hijacker of gastric epithelial cell signaling.Biochem Pharmacol. 2007; 73: 1697-1702Crossref PubMed Scopus (91) Google Scholar, and, if so, monoamine oxidases (MAOs), which generate the ROS mediator H2O2 and are a component of the mitochondrial membrane found in most cell types in the body,6Weyler W Hsu YP Breakefield XO Biochemistry and genetics of monoamine oxidase.Pharmacol Ther. 1990; 47: 391-417Crossref PubMed Scopus (290) Google Scholar might be expected to be one of the sources. Prolonged exposure to pathogenic bacteria and/or their secreted virulence factors may result in delayed wound closure and pathological tissue changes.7Robson MC Kucukcelebi A Carp SS Hayward PG Hui PS Cowan WT Ko F Cooper DM Maintenance of wound bacterial balance.Am J Surg. 1999; 178: 399-402Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarIn vitro, lipopolysaccharide (LPS) derived from either Pseudomonas aeruginosa or Escherichia coli strongly inhibits epithelial migration and is therefore implicated as a factor in the failure of wound closure.8Loryman C Mansbridge J Inhibition of keratinocyte migration by lipopolysaccharide.Wound Rep Reg. 2007; 16: 45-51Crossref Scopus (38) Google ScholarIn vivo, periodontal disease offers a model of such chronic wound conditions. The periodontal sulcus is located in a space between the tooth and surrounding gingival tissue and is composed of junctional epithelium, which interfaces with periodontal soft and mineralized tissue. The recess of the sulcus favors population by an adherent bacterial biofilm and hence chronic challenge by virulence factors. Conversion of this biofilm to a Gram-negative, LPS-rich microbial population is associated with the conversion of the junctional to pocket epithelium, which is regarded as an early marker of disease initiation.9Bosshardt DD Lang NP The junctional epithelium: from health to disease.J Dent Res. 2005; 84: 9-20Crossref PubMed Scopus (242) Google Scholar Periodontitis is associated with oxidative stress,10Akalın FA Baltacıoğlu E Alver A Karabulut E Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis.J Clin Periodontol. 2007; 34: 558-565Crossref PubMed Scopus (206) Google Scholar decreased total antioxidant status,11Chapple IL Brock GR Milward MR Ling N Matthews JB Compromised GCF total antioxidant capacity in periodontitis: cause or effect?.J Clin Periodontol. 2007; 34: 103-110Crossref PubMed Scopus (127) Google Scholar and/or increased lipid peroxidation in gingival crevicular fluid and saliva.12Tomofuji T Azuma T Kusano H Sanbe T Ekuni D Tamaki N Yamamoto T Watanabe T Oxidative damage of periodontal tissue in the rat periodontitis model: effects of a high-cholesterol diet.FEBS Lett. 2006; 580: 3601-3604Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Experimentally induced periodontitis provides a valuable model of chronic wounds and alveolar bone loss. Rats treated daily under anesthesia by application of E. coli LPS into the gingival sulcus showed disease as indicated by elongation of rete ridges and onset of apical migration of junctional epithelium at 8 weeks. Addition of Streptomyces griseus protease to the LPS treatment further potentiated the LPS-induced effects, yet protease treatment alone had no significant effect.13Ekuni D Yamamoto T Yamanaka R Tachibana K Watanabe T Proteases augment the effects of lipopolysaccharide in rat gingiva.J Periodont Res. 2003; 38: 591-596Crossref PubMed Scopus (32) Google Scholar The LPS/protease model also established that the progressive development of disease was associated with apoptosis of adjacent fibroblasts and destruction of collagen fibers relative to both sham-treated and untreated (time 0) controls.14Ekuni D Tomofuji T Yamanaka R Tachibana K Yamamoto T Watanabe T Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases.J Periodontol. 2005; 76: 43-48Crossref PubMed Scopus (42) Google Scholar This model also demonstrated that elevated local and plasma oxidative stress, as measured by 8-hydroxydeoxyguanosine levels, was associated with chronic wound formation and alveolar bone loss.15Ekuni D Tomofuji T Tamaki N Sanbe T Azuma T Yamanaka R Yamamoto T Watanabe T Mechanical stimulation of gingiva reduces plasma 8-OHdG level in rat periodontitis.Arch Oral Biol. 2008; 53: 324-329Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar In this study, the established LPS/protease-induced rat chronic wound model13Ekuni D Yamamoto T Yamanaka R Tachibana K Watanabe T Proteases augment the effects of lipopolysaccharide in rat gingiva.J Periodont Res. 2003; 38: 591-596Crossref PubMed Scopus (32) Google Scholar, 14Ekuni D Tomofuji T Yamanaka R Tachibana K Yamamoto T Watanabe T Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases.J Periodontol. 2005; 76: 43-48Crossref PubMed Scopus (42) Google Scholar, 15Ekuni D Tomofuji T Tamaki N Sanbe T Azuma T Yamanaka R Yamamoto T Watanabe T Mechanical stimulation of gingiva reduces plasma 8-OHdG level in rat periodontitis.Arch Oral Biol. 2008; 53: 324-329Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar was used to analyze epithelial ROS signaling leading to alveolar bone loss relative to time 0 controls. First, epithelial tissues isolated by laser-capture microdissection were analyzed by expression microarray for transcriptional changes associated with the onset of epithelial disease and alveolar bone loss. Second, because microdissected disease epithelia were shown to have elevated infiltration of polymorphonuclear leukocyte (PMN) immune cells, consistent with the inflammatory process, pro-oxidative genes of interest, monoamine oxidase A (Maoa) and B (Maob) were validated both by RT-quantitative (q) PCR and at the protein level (MAO/A and MAO/B) in cell culture. This validation was done using a confirmed PMN-free primary culture of porcine junctional epithelia, both because isolation of sufficient rat junctional epithelia for culture was impractical and to confirm the conservation of the mechanism across species. Third, because tumor necrosis factor-α (TNF-α) has been reported to be up-regulated in chronic wounds16Streit M Beleznay Z Braathen LR Topical application of the tumour necrosis factor-α antibody infliximab improves healing of chronic wounds.Int Wound J. 2006; 3: 171-179Crossref PubMed Scopus (80) Google Scholar and involved in bone loss,17Lam J Takeshita S Barker JE Kanagawa O Ross FP Teitelbaum SL TNF-α induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand.J Clin Invest. 2000; 106: 1481-1488Crossref PubMed Scopus (1115) Google Scholar LPS-associated ROS induction was assayed in association with elevated TNF-α protein expression both in vivo and in vitro. Fourth, the effects of LPS on TNF-α activity were further characterized in vitro using ROS inhibitors and MAO inhibitors. Finally, topically applied phenelzine, an MAO inhibitor, was tested in vivo for modulation of LPS-associated H2O2 production, TNF-α expression, PMN infiltration, systemic oxidative stress, and bone loss. Animal experiments complied with guidelines of and were approved by the Animal Research Committee of The University of British Columbia and the Animal Research Control Committee of Okayama University Dental School. A rat periodontitis model was used as described previously: Animals were anesthetized daily using Isoflurane (Baxter, Toronto, ON, Canada), and then 25 μg/μl of serotype O55:B5 E. coli LPS (Sigma-Aldrich, St. Louis, MO) with 2.25 U/μl of S. griseus type XIV proteases (Sigma-Aldrich), which was used for acceleration of LPS penetration, resuspended in pyrogen-free water (ICN Biomedical Inc., Aurora, OH), was introduced by micropipette into the left and right palatal gingival sulcus of all three maxillary molars as described previously.13Ekuni D Yamamoto T Yamanaka R Tachibana K Watanabe T Proteases augment the effects of lipopolysaccharide in rat gingiva.J Periodont Res. 2003; 38: 591-596Crossref PubMed Scopus (32) Google Scholar, 14Ekuni D Tomofuji T Yamanaka R Tachibana K Yamamoto T Watanabe T Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases.J Periodontol. 2005; 76: 43-48Crossref PubMed Scopus (42) Google Scholar A total of 14 male Wistar strain rats (6 weeks old) were used per experiment, with seven animals in each of the time 0 control and 8-week treatment groups. In another experiment using 21 male Wistar strain rats, 4 weeks after LPS/protease treatment was begun in 14 animals, this group were randomly divided into two groups of seven animals each. Thereafter, in addition to LPS/protease treatment, one group of rats also received topical application of 0.5 μl of 250 mg/ml phenelzine (Sigma-Aldrich) in pyrogen-free water for 4 weeks. The third group of 7 rats received topical application of pyrogen-free water for 8 weeks. Before death, rats were deeply anesthetized using Isoflurane and the right palatal gingival soft tissue was collected by sharp dissection, immediately embedded in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA), frozen in liquid nitrogen, and stored at −86°C until required. Sections from these blocks were subsequently used for laser dissection and processed for microarray analysis. After removal of the right palatal biopsy sample, rats were euthanized by intracardiac perfusion with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) under deep general anesthesia. After initial fixation, the left maxillary molar regions were resected en bloc from each rat. Tissues were decalcified with 10% tetrasodium-EDTA aqueous solution (pH 7.4) for 2 weeks at 4°C. Paraffin-embedded buccolingual 5-μm sections were made and used for morphometric and immunohistochemical analysis. Paraffin sections of 5-μm thickness were stained with H&E, and the distances from the cemento-enamel junction to the coronal aspect of the connective tissue attachment (epithelial migration) and from the cemento-enamel junction to the coronal margin of alveolar bone (bone loss) were measured using a microgrid at ×200 magnification. The number of PMNs was determined per 0.05 mm2 under a magnification of ×400.13Ekuni D Yamamoto T Yamanaka R Tachibana K Watanabe T Proteases augment the effects of lipopolysaccharide in rat gingiva.J Periodont Res. 2003; 38: 591-596Crossref PubMed Scopus (32) Google Scholar Means of histological data were calculated for each rat. Serial 8-μm-thick cryosections were cut on the day of use using a cryostat (Cambridge Instruments, Heidelberg, Germany) onto membrane slides (Zeiss P.A.L.M., Bernried, Germany) from three blocks each of the control and experimental groups. Immediately before use slides were briefly thawed, stained with Mayer’s hematoxylin (Sigma-Aldrich), rinsed with RNase-free H2O, and drained. Slides were sequentially dehydrated through 75, 95, and 100% ethanol for 30 seconds each, transferred into xylene for 5 minutes, and air-dried for 5 minutes. Slides were immediately used for laser-capture dissection (Zeiss P.A.L.M.). Sufficient sections were collected to yield a minimum of 2 × 106 μm2 of epithelia from each animal. Sections from each animal were pooled, and RNA was extracted using the RNeasy Micro Kit (Qiagen, Mississauga, ON, Canada). Amplified cRNA was produced and analyzed at Genome British Columbia (Vancouver, BC, Canada). Purified RNA was amplified using a GeneChip Expression 3′-Amplification Two-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA), cRNA quality/quantity was assayed using a lab-on-a-chip/Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), and cRNA was subject to expression analysis using a GeneChip Rat Genome 230 2.0 Array (Affymetrix). Microarray data were analyzed using principal component and dChip software.18Li C Automating dChip: toward reproducible sharing of microarray data analysis.BMC Bioinformatics. 2008; 9: 231Crossref PubMed Scopus (56) Google Scholar For RT-qPCR cell cultures were processed using an RNeasy Micro Kit as above. Template was synthesized using a SuperScript III First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA). Primers of interest (Table 1) were designed from porcine sequences using software from and synthesized by Integrated DNA Technologies (Coralville, IA). qPCR was performed using Platinum SYBR Green qPCR Super Mix-UDG (Invitrogen), and primers were subjected to 50°C for 10 minutes with initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 20 seconds. Raw data were analyzed with a relative quantification software tool (REST) using two internal reference genes (Gapdh and Actb), and results are presented as fold change relative to controls.19Pfaffl MW Horgan GW Dempfle L Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google ScholarTable 1Primer SequencesGeneAccessionSequenceMaoaNM_001001640F: 5′-AGGAACGGAAGTTTGTAGGCGGAT-3′R: 5′-ATAGGTGACAGGACACCTCAGCTT-3′MaobAY596820F: 5′-TTACAAAGAGCCCTTCTGGAGGCA-3′R: 5′-TGGAATCATCCAACGTGTAGGCGA-3′TnfaEU682384F: 5′-GCCCACGTTGTAGCCAATGTCAAA-3′R: 5′-GTTGTCTTTCAGCTTCACGCCGTT-3′GapdhAF017079F: 5′-GCAAAGTGGACATTGTCGCCATCA-3′R: 5′-CCGTGGAATTTGCCATGGGTAGAA-3′ActbDQ452569F: 5′-AGCTGGAGTCTTTCTCGTGTTTGC-3′R: 5′-TCACACGAGCCAGTGTTAGTACCT-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Representative cryosections or deparaffinized sections were stained with the primary antibodies recognizing MAO/A, MAO/B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and TNF-α (R&D Systems, Minneapolis, MN) in PBS/bovine serum albumin and Histofine Simple Stain MAX PO Kits (Nichirei Co., Tokyo, Japan). In brief, deparaffinized tissue sections or cryosections were immersed in methanol containing 0.3% H2O2 for 30 minutes to block endogenous peroxidase activity. The sections were treated with secondary antibody (Fab′)-conjugated peroxidase complex for 30 minutes. Peroxidase staining was performed for 5 to 10 minutes using a solution of 3,3′-diaminobenzidine tetrahydrochloride in 50 mmol/L Tris-HCl (pH 7.5) containing 0.001% H2O2. The sections were counterstained with Mayer’s hematoxylin. Digital quantification was performed to segment the area of interest and determine the ratio of positive expression in a given area using the Scion Image (Scion Corp., Frederick, MD) computer program as described previously.20Ekuni D Firth JD Putnins EE Regulation of epithelial cell growth factor receptor protein and gene expression using a rat periodontitis model.J Periodontal Res. 2006; 4: 340-349Crossref Scopus (10) Google Scholar Porcine ligament epithelial cells were isolated as described previously21Brunette DM Melcher AH Moe HK Culture and origin of epithelium-like and fibroblast-like cells from porcine periodontal ligament explants and cell suspensions.Arch Oral Biol. 1976; 21: 393-400Abstract Full Text PDF PubMed Scopus (156) Google Scholar and maintained in α-minimal essential medium (Gibco BRL, Rockville, MD) supplemented with 15% fetal bovine serum (PAA Laboratories Inc., Etobicoke, ON, Canada). Cells were plated into 96-well plates for RT-qPCR or H2O2 assay, 24-well plates for immunostaining or TNF-α assay, or 35-mm dishes for flow cytometry. When cultures were 75% confluent, the cells were quiesced overnight in serum-free α-minimal essential medium. Cells were treated with E. coli LPS (O55:B) at 12.5 to 1600 ng/ml. Long-term experiments of up to 2 weeks were performed with 2.5% serum and 250 ng/ml LPS. In some experiments, cells were pretreated with N-acetylcysteine (10 to 80 mmol/L, 1 hour) or catalase (125 to 1000 U/ml, 1 hour) (Sigma-Aldrich) and thereafter with or without 800 ng/ml LPS. In other experiments, cells were treated with the MAO/B inhibitor R-(−)-deprenyl hydrochloride or the MAO/A+B inhibitor phenelzine sulfate salt (Sigma-Aldrich) at 3 to 100 μmol/L in the absence or presence of 800 ng/ml LPS. Quantification of TNF-α levels in cell supernatant was performed using DuoSet enzyme-linked immunosorbent assay kits (R&D Systems). The 96-well microtiter plates were precoated with a monoclonal mouse anti-porcine TNF-α antibody. Then 200 ml of the samples was transferred to 96-well plates from 24-well plates, standards were applied and run in parallel, and all plates were incubated for 2 hours at room temperature. Plates were washed and further incubated with a biotinylated goat anti-porcine TNF-α antibody. The specifically bound antibody was detected by application of streptavidin-horseradish peroxidase. Peroxidase activity was visualized by the addition of tetramethylbenzidine substrate solution in H2O2. After 20 minutes of incubation, 2 N H2SO4 stop solution was added, and the plates were read at 450 nm. Semiconfluent, quiescent porcine ligament epithelial cultures were treated with increasing concentrations of LPS as above. Cells were recovered by trypsinization, neutralized with 10% serum, washed with PBS for flow cytometry or left in situ for immunostaining, and then fixed in 2% formaldehyde/5% sucrose for 1 hour at room temperature. Fixative was washed out three times with PBS, and then cells were blocked for 1 hour in hybridization buffer (3% bovine serum albumin, 0.2 mol/L glycine, and 0.1% saponin), which was also used in subsequent steps. Samples were incubated with MAO/A, MAO/B, or TNF-α antibody for 1 hour and then washed three times. Samples were incubated with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Eugene, OR) and finally were washed three times in PBS. Five groups of 10,000 cells from triplicate experiments were analyzed using a FACSCalibur system (BD Biosciences, San Jose, CA) or by epifluorescence and were digitally recorded. Samples stained with secondary antibody alone were included to standardize results relative to autofluorescence. Serum H2O2 levels were measured colorimetrically using the Amplex Red Kit (Molecular Probes). Fluorescence intensity was recorded with excitation in the range of 530 to 560 nm and emission of 590 nm. To directly detect ROS in cryosections or cultured cells, samples were incubated with 0.5 mg/ml 3,3-diaminobenzidine and 0.1 mol/L Tris-HCl, pH 7.6, alone (H2O2) or including 0.1 mg/ml MnCl2 (O·2)22Kerver ED Vogels IM Bosch KS Vreeling-Sindelarova H Van den Munckhof RJ Frederiks WM In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine.Histochem J. 1997; 29: 229-237Crossref PubMed Scopus (58) Google Scholar for 10 minutes and then were washed three times with PBS, counterstained with hematoxylin, washed three times with PBS again, fixed (2% paraformaldehyde, 5% sucrose, and PBS), and mounted. Images were digitally recorded under tungsten illumination and quantified as above. ROS scavenging was also tested cell-free with increasing concentrations of catalase, R-(−)-deprenyl hydrochloride or phenelzine sulfate incubated with H2O2 (1 mmol/L, room temperature, 1 hour) and then incubated with 3,3′-diaminobenzidine tetrahydrochloride as above and measured spectrophotometrically at 560 nm. Data analysis was performed using a statistical software package (SPSS 15.0J for Windows, SPSS Japan, Tokyo, Japan). Data are expressed as means ± SD. Statistical analysis was performed using a Mann-Whitney U-test for comparison between the two groups (P < 0.05). Differences among the three groups were analyzed using a Kruskal-Wallis test followed by a Mann-Whitney U-test, using the Bonferroni correction to adjust probability (P < 0.05/3 = 0.017). The previously established experimental periodontitis model was used with time 0 controls (healthy) and rats that received 8 weeks of daily applications of LPS/protease combined treatment to the gingival sulcus (disease).13Ekuni D Yamamoto T Yamanaka R Tachibana K Watanabe T Proteases augment the effects of lipopolysaccharide in rat gingiva.J Periodont Res. 2003; 38: 591-596Crossref PubMed Scopus (32) Google Scholar, 14Ekuni D Tomofuji T Yamanaka R Tachibana K Yamamoto T Watanabe T Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases.J Periodontol. 2005; 76: 43-48Crossref PubMed Scopus (42) Google Scholar The treated group exhibited apical migration of junctional epithelium characteristic of disease (Figure 1, A and D). As confirmed by morphology, cryosections from healthy (Figure 1, B and C) and diseased (Figure 1, E and F) rats were processed by laser-capture microscopy for the isolation of epithelium. Laser captures sections of about 2 million μm2 of epithelium from each group were pooled, RNA was purified, and then sections were subjected to two rounds of amplification and cRNA synthesis (Figure 2). Far fewer sections were required from the disease group to reach the required total area because of increased epithelial proliferation associated with LPS treatment as shown in Figure 1. This response to bacterial challenge may be similar to the increased proliferation reported in gastric mucosal epithelia in response to H. pylori infections.23Lynch DAF Mapstone NP Clarke AMT Sobala GM Jackson P Morrison L Dixon MF Quirke P Axon ATR Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy.Gut. 1995; 36: 346-350Crossref PubMed Scopus (171) Google Scholar Total amplified cRNA and cRNA per unit area were highly consistent between healthy (control group) and diseased (8-week treatment group) animals (Figure 2A), and RNA yields from healthy (Figure 2B) and diseased (Figure 2C) rats showed consistent size profiles that ranged from 52 to 3891 nucleotides with a median size of 510 nucleotides for all samples, indicating that LPS treatment was not associated with increased RNA degradation.Figure 2cRNA amplification of laser-capture microdissected healthy and disease-associated epithelia. Palatal biopsy samples from control and 8-week LPS-treated animals (n = 3/group) were removed under general anesthesia, embedded in optimal cutting temperature compound, and frozen. Serial 8-μm cryosections were cut onto membrane slides, dehydrated, and processed for laser-capture microdissection. Sections from each animal were pooled, and total RNA was amplified through two cycles. Final cRNA was quantified and expressed per unit area of epithelia (A). Amplified cRNA from control (B) and 8-week treated (C) animals was compared for cRNA relative quality, cRNA size range (52 to 3891 nucleotides), and median size (510 nucleotides) for all groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gene expression changes were assayed by subjecting cRNA from healthy and diseased animals to microarray analysis. Normalized data were first analyzed by principal component analysis, which showed that healthy and diseased samples grouped quite separately, but closely, within treatment groups, although one disease sample, number 2 rat in the 8 weeks of treatment group (8W2ep), was deemed to be somewhat of an outlier and was excluded from further analysis (Figure 3). Of particular interest, on the basis of ranked change, it was found that of the genes induced in response to disease onset (9031 of 19,730) only a very small number (42) showed a greater than fourfold increase in expression (Table 2). However, within the top 10 of this group, three genes, monoamine oxidase B (Maob, 5.72-fold) and flavin-containing monooxygenase 1 (Fmo1, 6.70-fold) and 2 (Fmo2, 7.26-fold) are involved in reactive oxygen signaling (Table 2). Further querying of the databa
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